This document summarizes the discovery of duplicated VegfA and KDR receptor genes in zebrafish that mediate vascular development. Specifically:
- The researchers identified a duplicated zebrafish VegfA gene (VegfAb) that encodes 171- and 210-amino acid isoforms not found in the single VegfA gene.
- They also found a duplicated KDR receptor gene (Kdrb) that encodes a receptor similar to mammalian KDR.
- Knockdown experiments in zebrafish showed that both VegfAb and the duplicated KDR receptor genes play important roles in vascular development.
- Further experiments demonstrated that the VegfAb isoforms are poorly secreted compared to VegfA isoforms
1) The document summarizes research into genetic interactors of the BRCA2 tumor suppressor gene, which is linked to hereditary breast cancer. A retroviral screening identified BRE as a genetic interactor that rescues lethality in BRCA2-deficient cells.
2) Further experiments showed that BRE overexpression in BRCA2-deficient cells leads to increased levels of the Cdc25A cell cycle regulator after DNA damage, preventing cell cycle arrest.
3) BRE was found to interact with the transcription factor ATF3 and induce transcription of Cdc25A. Reporter assays aim to identify the role of ATF3 binding sites on the Cdc25A promoter in regulating its transcription
Derivation and functional characterization of distinct dc subsetsOfer Wellisch
1) Mouse hematopoietic stem cells were conditionally immortalized using a Hox oncoprotein to derive distinct dendritic cell (DC) subsets for analysis.
2) The method derived conventional DCs (cDCs) and plasmacytoid DCs (pDCs) that could be terminally differentiated in culture and analyzed functionally.
3) Preliminary results showed constitutive expression of antiviral genes in pDCs but not cDCs, suggesting an interferon autocrine loop in pDCs.
In vitro transcription and transfection of HCV genomic repliconBinodGupta27
ABSTRACT:
Introduction: Hepatitis C virus (HCV) is a positive stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Aims & Objectives: The study was conducted to establish the transfection of Huh 7.5 derived cell lines with In-vitro transcript of HCV pF6/JFH-1 for production of infectious virus particles in naïve Huh 7.5 cells, its detection by RT-PCR. Materials and Method: Huh 7.5 cells, a highly permissive cell lines for HCV replication, were grown in Dulbecco’s Modified Eagle’s Medium and pFL-J6/JFH plasmid was linearized with XbaI and subjected to in-vitro transcription using MEGAscript Kit (Ambion, USA) Huh-7.5 cells were transfected with 2.5 μg transcript using Lipofectamine 2000 transfection reagent (Invitrogen, USA) . The culture supernatant was collected after 24, 48 and 72 hr after incubation in fresh media and viral RNAs were isolated from it using Trizol LS reagent (Ambion, USA) and quantified by real-time quantitative RT-PCR. Total RNA was extracted from cells using Trizol reagent (Ambion, USA) and then RNA was subjected to cDNA synthesis using RevertAid reverse transcription (Thermo Fisher Scientific, USA). The PCR products were resolved by electrophoresis in 1.5% (w/v) agarose gels and images were captured by a Chemidoc XRS system (Bio-Rad, USA). Results: We observed Huh7.5 cells were cultured in DMEM. Plasmid FL-J6/JFH1 was linearized with the restriction enzyme XbaI and HCV RNA was obtained by In-vitro transcription and was transfected to grown Huh 7.5 cells shown by band on agarose gel and total RNA isolated after 24 hours of post infection followed by RT-PCR gave distinct band on gel whereas 48 and 72 hr did not. Infection of Huh 7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript gave a distinct band. This will help in understanding entire viral life cycle and its non-structural gene products like NS4B and NS5A that enhance the replicative capacity of replicons in Huh 7.5 cell lines for development of drug and vaccines.
A family of acetylcholine-gated chloride channel subunits in Caenorhabditis e...Igor Putrenko
This document summarizes the cloning and characterization of acetylcholine-gated chloride channel subunits in Caenorhabditis elegans. The authors cloned four subunits, ACC-1, ACC-2, ACC-3, and ACC-4, which form a distinct clade of ligand-gated ion channel genes in C. elegans. They found that ACC-1 and ACC-2 form homomeric channels that are gated by acetylcholine but not nicotine, blocked by D-tubocurarine but not α-bungarotoxin, and conduct chloride ions. ACC-3 and ACC-4 may interact with ACC-1 and ACC-2. This study identifies the first molecular
This study investigated the role of the DNA damage checkpoint kinase Chk1 in sister chromatid cohesion (SCC) and genome stability in Saccharomyces cerevisiae. The results showed that unlike SCC mutants, loss of CHK1 did not increase spontaneous or damage-induced allelic recombination or aneuploidy. Exposure of G2-arrested cells to ionizing radiation also did not increase allelic recombination or reduce survival in chk1 mutant cells as seen in SCC mutants. This suggests that Chk1 has a redundant role in controlling damage-induced SCC or that damage-induced SCC is redundant for maintaining genome stability in yeast.
This proposal aims to develop an assay to screen for inhibitors of West Nile virus (WNV) protease. The assay will utilize a Gal4 fusion system where the WNV protease is inserted between the Gal4 DNA binding and activation domains. Protease cleavage of the fusion would prevent GFP expression, while inhibition would allow GFP expression. Preliminary data shows the mutated, non-cleavable fusion induces GFP, while the wild type fusion does not, demonstrating proof of concept. Stable cell lines will be generated expressing all assay components to enable high-throughput screening for WNV protease inhibitors.
Gene therapy aims to cure β-thalassemias by using lentiviral vectors to insert functional β-globin genes into hematopoietic stem cells. The first patient treated achieved long-term transfusion independence with stable multi-year expression of the corrected globin. Analysis found most genetically modified cells contained the vector integrated near the HMGA2 gene, though the majority of cells remained unmodified. Ongoing work continues to optimize the therapy.
Cord blood is a naturally discarded tissue that contains a high proportion of circulating hematopoietic stem cells. It allows HLA mismatch transplantation, providing cure to hematological malignancies with an attenuated risk of severe graft-versus-host disease through a graft-versus-leukemia effect. Cord blood transplantation is unique in its immunogenetics profile, preserving the immune cells carried in the cord blood graft, including T-regulatory cells and natural killer cells. Early reconstitution of lymphocytes from cord blood grafts predicts better outcomes. Advances in cord blood transplantation aim to take advantage of its immunological properties through donor selection accounting for HLA matching, maternal antigens, and cell dose to maximize graft-versus-leuke
1) The document summarizes research into genetic interactors of the BRCA2 tumor suppressor gene, which is linked to hereditary breast cancer. A retroviral screening identified BRE as a genetic interactor that rescues lethality in BRCA2-deficient cells.
2) Further experiments showed that BRE overexpression in BRCA2-deficient cells leads to increased levels of the Cdc25A cell cycle regulator after DNA damage, preventing cell cycle arrest.
3) BRE was found to interact with the transcription factor ATF3 and induce transcription of Cdc25A. Reporter assays aim to identify the role of ATF3 binding sites on the Cdc25A promoter in regulating its transcription
Derivation and functional characterization of distinct dc subsetsOfer Wellisch
1) Mouse hematopoietic stem cells were conditionally immortalized using a Hox oncoprotein to derive distinct dendritic cell (DC) subsets for analysis.
2) The method derived conventional DCs (cDCs) and plasmacytoid DCs (pDCs) that could be terminally differentiated in culture and analyzed functionally.
3) Preliminary results showed constitutive expression of antiviral genes in pDCs but not cDCs, suggesting an interferon autocrine loop in pDCs.
In vitro transcription and transfection of HCV genomic repliconBinodGupta27
ABSTRACT:
Introduction: Hepatitis C virus (HCV) is a positive stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Aims & Objectives: The study was conducted to establish the transfection of Huh 7.5 derived cell lines with In-vitro transcript of HCV pF6/JFH-1 for production of infectious virus particles in naïve Huh 7.5 cells, its detection by RT-PCR. Materials and Method: Huh 7.5 cells, a highly permissive cell lines for HCV replication, were grown in Dulbecco’s Modified Eagle’s Medium and pFL-J6/JFH plasmid was linearized with XbaI and subjected to in-vitro transcription using MEGAscript Kit (Ambion, USA) Huh-7.5 cells were transfected with 2.5 μg transcript using Lipofectamine 2000 transfection reagent (Invitrogen, USA) . The culture supernatant was collected after 24, 48 and 72 hr after incubation in fresh media and viral RNAs were isolated from it using Trizol LS reagent (Ambion, USA) and quantified by real-time quantitative RT-PCR. Total RNA was extracted from cells using Trizol reagent (Ambion, USA) and then RNA was subjected to cDNA synthesis using RevertAid reverse transcription (Thermo Fisher Scientific, USA). The PCR products were resolved by electrophoresis in 1.5% (w/v) agarose gels and images were captured by a Chemidoc XRS system (Bio-Rad, USA). Results: We observed Huh7.5 cells were cultured in DMEM. Plasmid FL-J6/JFH1 was linearized with the restriction enzyme XbaI and HCV RNA was obtained by In-vitro transcription and was transfected to grown Huh 7.5 cells shown by band on agarose gel and total RNA isolated after 24 hours of post infection followed by RT-PCR gave distinct band on gel whereas 48 and 72 hr did not. Infection of Huh 7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript gave a distinct band. This will help in understanding entire viral life cycle and its non-structural gene products like NS4B and NS5A that enhance the replicative capacity of replicons in Huh 7.5 cell lines for development of drug and vaccines.
A family of acetylcholine-gated chloride channel subunits in Caenorhabditis e...Igor Putrenko
This document summarizes the cloning and characterization of acetylcholine-gated chloride channel subunits in Caenorhabditis elegans. The authors cloned four subunits, ACC-1, ACC-2, ACC-3, and ACC-4, which form a distinct clade of ligand-gated ion channel genes in C. elegans. They found that ACC-1 and ACC-2 form homomeric channels that are gated by acetylcholine but not nicotine, blocked by D-tubocurarine but not α-bungarotoxin, and conduct chloride ions. ACC-3 and ACC-4 may interact with ACC-1 and ACC-2. This study identifies the first molecular
This study investigated the role of the DNA damage checkpoint kinase Chk1 in sister chromatid cohesion (SCC) and genome stability in Saccharomyces cerevisiae. The results showed that unlike SCC mutants, loss of CHK1 did not increase spontaneous or damage-induced allelic recombination or aneuploidy. Exposure of G2-arrested cells to ionizing radiation also did not increase allelic recombination or reduce survival in chk1 mutant cells as seen in SCC mutants. This suggests that Chk1 has a redundant role in controlling damage-induced SCC or that damage-induced SCC is redundant for maintaining genome stability in yeast.
This proposal aims to develop an assay to screen for inhibitors of West Nile virus (WNV) protease. The assay will utilize a Gal4 fusion system where the WNV protease is inserted between the Gal4 DNA binding and activation domains. Protease cleavage of the fusion would prevent GFP expression, while inhibition would allow GFP expression. Preliminary data shows the mutated, non-cleavable fusion induces GFP, while the wild type fusion does not, demonstrating proof of concept. Stable cell lines will be generated expressing all assay components to enable high-throughput screening for WNV protease inhibitors.
Gene therapy aims to cure β-thalassemias by using lentiviral vectors to insert functional β-globin genes into hematopoietic stem cells. The first patient treated achieved long-term transfusion independence with stable multi-year expression of the corrected globin. Analysis found most genetically modified cells contained the vector integrated near the HMGA2 gene, though the majority of cells remained unmodified. Ongoing work continues to optimize the therapy.
Cord blood is a naturally discarded tissue that contains a high proportion of circulating hematopoietic stem cells. It allows HLA mismatch transplantation, providing cure to hematological malignancies with an attenuated risk of severe graft-versus-host disease through a graft-versus-leukemia effect. Cord blood transplantation is unique in its immunogenetics profile, preserving the immune cells carried in the cord blood graft, including T-regulatory cells and natural killer cells. Early reconstitution of lymphocytes from cord blood grafts predicts better outcomes. Advances in cord blood transplantation aim to take advantage of its immunological properties through donor selection accounting for HLA matching, maternal antigens, and cell dose to maximize graft-versus-leuke
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
This study investigated genetic variations among four sheep breeds related to fecundity and weight gain using microsatellite markers. Blood samples were collected from 40 female sheep from four breeds and were used to extract DNA. Five microsatellites linked to genes for litter size (Fec gene) and fat deposition/weight gain (Ob gene) were selected. Polymerase chain reaction and automated sequencing were used to analyze polymorphisms in the microsatellite markers. Serum was also isolated from blood samples and tested via ELISA to measure levels of LH and leptin hormones. The aim was to analyze the effects of genetic differences among the breeds for two traits: fecundity and weight gain.
Derivation of highly enriched cultures of differentiated cells from human par...Nikolay Turovets
California Institute for Regenerative Medicine (CIRM) & Medical Research Council (MRC)
Human SCNT Workshop.
14 June, 2010, San Francisco, CA
Workshop report: http://www.cirm.ca.gov/files/PDFs/Publications/Human_SCNT_Workshop_Report.pdf
1) The study analyzed the effects of Csk knockouts on development of the initial segment of the mouse epididymis. Csk knockout was expected to promote cell proliferation and differentiation through increased ERK pathway activity due to lack of inhibition of SRC kinases.
2) A tissue-specific Csk conditional knockout mouse model was generated using Cre/lox recombination. Genotyping identified one mouse with the desired genotype.
3) Preliminary results found increased vasculogenesis in the initial segment of Csk knockout mice, suggesting effects on differentiation through the ERK pathway. Immunofluorescence found decreased activity of phospho-SRC in knockouts while other markers were similar to controls.
Researchers investigated the roles of two sirtuin genes, SIRT4 and SIRT7, in grapevine physiology. In vitro assays showed both genes exhibited very weak NAD+-dependent deacetylase activity, even in the presence of resveratrol. Long fragments of the true coding sequences for SIRT4 and SIRT7 were obtained via RT-PCR that highly corresponded to hypothetical sequences. Basal transcription of both genes was found in grape cell cultures. Exposure to methyl jasmonate and UV-C radiation showed no influence on SIRT4 expression, while SIRT7 expression was more complex and non-linear, requiring further study. The work provides a starting point for understanding
This document contains a sample exam for the GATE biotechnology exam from 2000. It includes 31 multiple choice or short answer questions testing knowledge of topics like restriction enzymes, DNA structure, gene expression, cloning, PCR, and more. The questions range in difficulty from recalling basic concepts to applying knowledge through calculations.
This document summarizes a test that analyzes mutations in the kinase domain of the BCR-ABL1 gene in patients with chronic myeloid leukemia (CML) or Philadelphia-chromosome positive acute lymphoblastic leukemia (ALL). It detects over 99% of reported mutations using reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Certain mutations are associated with resistance to specific tyrosine kinase inhibitors used to treat CML and ALL. Identifying mutations can help guide treatment decisions. The test is recommended for patients who show an inadequate response or loss of response to tyrosine kinase inhibitors according to NCCN guidelines.
This study uses allele-specific chromosome conformation capture sequencing (4C-seq) to compare the chromatin configuration and genomic contacts of the productive and non-productive immunoglobulin heavy chain (IgH) alleles in mature B cells. The study finds that despite being physically and functionally different, the productive and non-productive IgH alleles in B cells share many chromosomal contacts and largely reside in active chromatin, unlike the IgH locus in brain cells which resides in repressive chromatin. Additionally, in mature B cells the distal VH regions of both IgH alleles position themselves away from active chromatin, which may help restrict enhancer activity to the productively rearranged VH promoter.
This document reports on a study examining the role of platelet-derived growth factor (PDGF) autocrine loops in human astrocytoma cells. The study shows that dominant-negative mutants of PDGF that disrupt PDGF ligand formation are able to revert the transformed phenotype of PDGF-transformed BALB/c 3T3 cells and two independent human astrocytoma cell lines. In contrast, the mutants did not alter the growth of cell lines transformed by other oncogenes or of other human cancer cell lines. These results support the view that PDGF autocrine loops contribute to the transformed phenotype of at least some human astrocytomas.
Transplantation in sensitized patients(seminar)Vishal Golay
This document discusses HLA typing, crossmatching, and transplantation in sensitized patients. It begins with a brief history of organ transplantation. It then covers topics such as the structure and function of MHC molecules, methods of HLA typing including serological and DNA-based techniques, interpreting HLA typing reports, detecting sensitization through antibody detection tests, defining and identifying sensitized patients, and challenges in transplanting sensitized patients. Advances in diagnostics and therapeutics have helped increase transplantation options for sensitized patients.
This document describes research aimed at identifying a transcription factor that links activation of the synapsis checkpoint during meiosis in C. elegans to the apoptotic pathway. The researcher performed an RNAi screen of 18 candidate transcription factors in a synapsis checkpoint mutant background. This identified nhr-84 as a potential factor, as its knockdown reduced apoptosis levels. Further experiments showed that nhr-84 knockdown only reduced apoptosis in checkpoint mutants, not in wild-type, indicating it specifically promotes apoptosis in response to synapsis checkpoint activation. This suggests nhr-84 may be the transcription factor that links the synapsis checkpoint to apoptosis.
This document summarizes a student research project that analyzed the frequency of the CCR5 Delta 32 allele in a population in Northeastern Ohio. The students mapped the CCR5 gene, developed a DNA collection and analysis protocol, and tested 50 samples. Their results found that 5 out of the 50 samples were heterozygous for the Delta 32 allele, indicating a gene frequency of 5% in the population. The students propose continuing their research and exploring using the Delta 32 mutation for potential gene therapy applications.
This document summarizes a student research project that analyzed the frequency of the CCR5 Δ32 allele in a population in Northeastern Ohio. The students mapped primers for the CCR5 gene, developed a DNA collection and analysis protocol, and tested 50 samples from their population. Their results found that 45 samples were homozygous wild-type, 5 were heterozygous for the Δ32 mutation, and none were homozygous. They conclude that the Δ32 allele frequency in this population is 5%. Future plans include further testing at their college and using the Δ32 mutation for potential gene therapy.
This document reports on a study that found adult hippocampal neural stem and progenitor cells (NSPCs) secrete significant amounts of vascular endothelial growth factor (VEGF), which plays an important role in maintaining the adult neurogenic niche. The study showed that NSPCs in the adult hippocampus express VEGF both in vivo and in vitro. Inducible knockout of VEGF specifically in NSPCs led to a 20-30% reduction in total hippocampal VEGF levels and impaired stem cell maintenance over time, demonstrating the functional relevance of NSPC-derived VEGF. These findings reveal that NSPCs act as a previously unknown secretory cell type that helps regulate the neurogenic niche through VEGF secretion.
This document summarizes a student project aimed at developing a gene therapy technique using CCR5 Δ32 mutation to potentially cure HIV/AIDS. The project involves collecting a blood sample from a participant with the CCR5 Δ32 mutation, purifying the DNA, inserting it into a vector, and transducing cells to integrate the modified gene and block HIV entry. The goal is to develop an easier gene therapy method that could cure HIV by reversing the virus's effects, as was seen in the case of Timothy Ray Brown.
This document summarizes research on the disruption of the cyclin D/CDK/INK4/Rb regulatory pathway in human neuroblastoma cell lines. The researchers found that 17 neuroblastoma cell lines highly expressed the CDK inhibitors p16INK4a and p18INK4c, but CDK6 kinase activity and phosphorylated Rb were still detected. One cell line was found to have a mutation in CDK6 that disrupts p16INK4a binding and prevents its inhibition of CDK6, bypassing the cell cycle block. The mechanisms allowing CDK6 activity in the other 16 cell lines despite high p16INK4a levels is unknown.
Efficient transformation of lactococcus lactis il1403 and generation of knock...CAS0609
This document describes an optimized protocol for efficiently transforming Lactococcus lactis IL1403 by electroporation. Key aspects of the protocol include growing cells in media supplemented with glycine and sucrose, harvesting them at mid-late log phase, washing them in buffers containing sucrose and EDTA, and electroporating them with a resistor in series. The utility of the protocol was demonstrated by generating single and double gene knock-out mutants using non-replicating vectors. Transformation efficiencies as high as 106 cfu/μg of DNA were achieved, allowing for genetic manipulation of L. lactis IL1403.
This document discusses how nicotine exposure may increase the risk of wet age-related macular degeneration (AMD) through its effects on the retinal pigment epithelium (RPE). The study finds that nicotine increases the ratio of pro-angiogenic vascular endothelial growth factor (VEGF) to anti-angiogenic pigment epithelium-derived factor (PEDF) produced by the RPE, possibly through activation of nicotinic acetylcholine receptors. This shift in growth factors may promote choroidal neovascularization, a key event in wet AMD. The results suggest nicotine exposure from secondhand smoke could play a role in the progression to wet AMD in passive smokers.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
This study investigated genetic variations among four sheep breeds related to fecundity and weight gain using microsatellite markers. Blood samples were collected from 40 female sheep from four breeds and were used to extract DNA. Five microsatellites linked to genes for litter size (Fec gene) and fat deposition/weight gain (Ob gene) were selected. Polymerase chain reaction and automated sequencing were used to analyze polymorphisms in the microsatellite markers. Serum was also isolated from blood samples and tested via ELISA to measure levels of LH and leptin hormones. The aim was to analyze the effects of genetic differences among the breeds for two traits: fecundity and weight gain.
Derivation of highly enriched cultures of differentiated cells from human par...Nikolay Turovets
California Institute for Regenerative Medicine (CIRM) & Medical Research Council (MRC)
Human SCNT Workshop.
14 June, 2010, San Francisco, CA
Workshop report: http://www.cirm.ca.gov/files/PDFs/Publications/Human_SCNT_Workshop_Report.pdf
1) The study analyzed the effects of Csk knockouts on development of the initial segment of the mouse epididymis. Csk knockout was expected to promote cell proliferation and differentiation through increased ERK pathway activity due to lack of inhibition of SRC kinases.
2) A tissue-specific Csk conditional knockout mouse model was generated using Cre/lox recombination. Genotyping identified one mouse with the desired genotype.
3) Preliminary results found increased vasculogenesis in the initial segment of Csk knockout mice, suggesting effects on differentiation through the ERK pathway. Immunofluorescence found decreased activity of phospho-SRC in knockouts while other markers were similar to controls.
Researchers investigated the roles of two sirtuin genes, SIRT4 and SIRT7, in grapevine physiology. In vitro assays showed both genes exhibited very weak NAD+-dependent deacetylase activity, even in the presence of resveratrol. Long fragments of the true coding sequences for SIRT4 and SIRT7 were obtained via RT-PCR that highly corresponded to hypothetical sequences. Basal transcription of both genes was found in grape cell cultures. Exposure to methyl jasmonate and UV-C radiation showed no influence on SIRT4 expression, while SIRT7 expression was more complex and non-linear, requiring further study. The work provides a starting point for understanding
This document contains a sample exam for the GATE biotechnology exam from 2000. It includes 31 multiple choice or short answer questions testing knowledge of topics like restriction enzymes, DNA structure, gene expression, cloning, PCR, and more. The questions range in difficulty from recalling basic concepts to applying knowledge through calculations.
This document summarizes a test that analyzes mutations in the kinase domain of the BCR-ABL1 gene in patients with chronic myeloid leukemia (CML) or Philadelphia-chromosome positive acute lymphoblastic leukemia (ALL). It detects over 99% of reported mutations using reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Certain mutations are associated with resistance to specific tyrosine kinase inhibitors used to treat CML and ALL. Identifying mutations can help guide treatment decisions. The test is recommended for patients who show an inadequate response or loss of response to tyrosine kinase inhibitors according to NCCN guidelines.
This study uses allele-specific chromosome conformation capture sequencing (4C-seq) to compare the chromatin configuration and genomic contacts of the productive and non-productive immunoglobulin heavy chain (IgH) alleles in mature B cells. The study finds that despite being physically and functionally different, the productive and non-productive IgH alleles in B cells share many chromosomal contacts and largely reside in active chromatin, unlike the IgH locus in brain cells which resides in repressive chromatin. Additionally, in mature B cells the distal VH regions of both IgH alleles position themselves away from active chromatin, which may help restrict enhancer activity to the productively rearranged VH promoter.
This document reports on a study examining the role of platelet-derived growth factor (PDGF) autocrine loops in human astrocytoma cells. The study shows that dominant-negative mutants of PDGF that disrupt PDGF ligand formation are able to revert the transformed phenotype of PDGF-transformed BALB/c 3T3 cells and two independent human astrocytoma cell lines. In contrast, the mutants did not alter the growth of cell lines transformed by other oncogenes or of other human cancer cell lines. These results support the view that PDGF autocrine loops contribute to the transformed phenotype of at least some human astrocytomas.
Transplantation in sensitized patients(seminar)Vishal Golay
This document discusses HLA typing, crossmatching, and transplantation in sensitized patients. It begins with a brief history of organ transplantation. It then covers topics such as the structure and function of MHC molecules, methods of HLA typing including serological and DNA-based techniques, interpreting HLA typing reports, detecting sensitization through antibody detection tests, defining and identifying sensitized patients, and challenges in transplanting sensitized patients. Advances in diagnostics and therapeutics have helped increase transplantation options for sensitized patients.
This document describes research aimed at identifying a transcription factor that links activation of the synapsis checkpoint during meiosis in C. elegans to the apoptotic pathway. The researcher performed an RNAi screen of 18 candidate transcription factors in a synapsis checkpoint mutant background. This identified nhr-84 as a potential factor, as its knockdown reduced apoptosis levels. Further experiments showed that nhr-84 knockdown only reduced apoptosis in checkpoint mutants, not in wild-type, indicating it specifically promotes apoptosis in response to synapsis checkpoint activation. This suggests nhr-84 may be the transcription factor that links the synapsis checkpoint to apoptosis.
This document summarizes a student research project that analyzed the frequency of the CCR5 Delta 32 allele in a population in Northeastern Ohio. The students mapped the CCR5 gene, developed a DNA collection and analysis protocol, and tested 50 samples. Their results found that 5 out of the 50 samples were heterozygous for the Delta 32 allele, indicating a gene frequency of 5% in the population. The students propose continuing their research and exploring using the Delta 32 mutation for potential gene therapy applications.
This document summarizes a student research project that analyzed the frequency of the CCR5 Δ32 allele in a population in Northeastern Ohio. The students mapped primers for the CCR5 gene, developed a DNA collection and analysis protocol, and tested 50 samples from their population. Their results found that 45 samples were homozygous wild-type, 5 were heterozygous for the Δ32 mutation, and none were homozygous. They conclude that the Δ32 allele frequency in this population is 5%. Future plans include further testing at their college and using the Δ32 mutation for potential gene therapy.
This document reports on a study that found adult hippocampal neural stem and progenitor cells (NSPCs) secrete significant amounts of vascular endothelial growth factor (VEGF), which plays an important role in maintaining the adult neurogenic niche. The study showed that NSPCs in the adult hippocampus express VEGF both in vivo and in vitro. Inducible knockout of VEGF specifically in NSPCs led to a 20-30% reduction in total hippocampal VEGF levels and impaired stem cell maintenance over time, demonstrating the functional relevance of NSPC-derived VEGF. These findings reveal that NSPCs act as a previously unknown secretory cell type that helps regulate the neurogenic niche through VEGF secretion.
This document summarizes a student project aimed at developing a gene therapy technique using CCR5 Δ32 mutation to potentially cure HIV/AIDS. The project involves collecting a blood sample from a participant with the CCR5 Δ32 mutation, purifying the DNA, inserting it into a vector, and transducing cells to integrate the modified gene and block HIV entry. The goal is to develop an easier gene therapy method that could cure HIV by reversing the virus's effects, as was seen in the case of Timothy Ray Brown.
This document summarizes research on the disruption of the cyclin D/CDK/INK4/Rb regulatory pathway in human neuroblastoma cell lines. The researchers found that 17 neuroblastoma cell lines highly expressed the CDK inhibitors p16INK4a and p18INK4c, but CDK6 kinase activity and phosphorylated Rb were still detected. One cell line was found to have a mutation in CDK6 that disrupts p16INK4a binding and prevents its inhibition of CDK6, bypassing the cell cycle block. The mechanisms allowing CDK6 activity in the other 16 cell lines despite high p16INK4a levels is unknown.
Efficient transformation of lactococcus lactis il1403 and generation of knock...CAS0609
This document describes an optimized protocol for efficiently transforming Lactococcus lactis IL1403 by electroporation. Key aspects of the protocol include growing cells in media supplemented with glycine and sucrose, harvesting them at mid-late log phase, washing them in buffers containing sucrose and EDTA, and electroporating them with a resistor in series. The utility of the protocol was demonstrated by generating single and double gene knock-out mutants using non-replicating vectors. Transformation efficiencies as high as 106 cfu/μg of DNA were achieved, allowing for genetic manipulation of L. lactis IL1403.
This document discusses how nicotine exposure may increase the risk of wet age-related macular degeneration (AMD) through its effects on the retinal pigment epithelium (RPE). The study finds that nicotine increases the ratio of pro-angiogenic vascular endothelial growth factor (VEGF) to anti-angiogenic pigment epithelium-derived factor (PEDF) produced by the RPE, possibly through activation of nicotinic acetylcholine receptors. This shift in growth factors may promote choroidal neovascularization, a key event in wet AMD. The results suggest nicotine exposure from secondhand smoke could play a role in the progression to wet AMD in passive smokers.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Complement and coagulation 2014 Captsone (formatted)Matt Gerace
1) Shiga toxin-producing E. coli infection can cause hemolytic uremic syndrome (HUS), a thrombotic microangiopathy characterized by low platelets, anemia, and kidney failure.
2) Shiga toxin damages endothelial cells, causing them to express tissue factor and cytokines that promote coagulation, leading to microvascular thrombosis.
3) Complement system activation, especially via the alternative pathway, is amplified by endothelial damage and augments the prothrombotic and inflammatory response, worsening the pathogenesis of HUS.
Biomaterials in oral and maxillofacial surgery /oral surgery courses by ...Indian dental academy
This document provides information about a seminar on biomaterials in oral and maxillofacial surgery presented by Dr. M. Prudhvi Raj. It discusses the introduction and definitions of biomaterials, selection criteria for biomedical materials, and common classes of biomaterials including metals, polymers, and ceramics. Specifically, it describes commonly used metallic biomaterials such as stainless steel, cobalt-chromium alloys, and titanium alloys; highlighting their compositions and properties.
A dental implant (also known as an endosseous implant or fixture) is a surgical component that interfaces with the bone of the jaw or skull to support a dental prosthesis such as a crown, bridge, denture, facial prosthesis or to act as an orthodontic anchor. The basis for modern dental implants is a biologic process called osseointegration where materials, such as titanium, form an intimate bond to bone.
We are going to discuss the four Biomaterials and their Dental Applications, the Advantages and Disadvantages of each Material.
Indian Dental Academy: will be one of the most relevant and exciting training
center with best faculty and flexible training programs for dental
professionals who wish to advance in their dental practice,Offers certified
courses in Dental implants,Orthodontics,Endodontics,Cosmetic Dentistry,
Prosthetic Dentistry, Periodontics and General Dentistry.
Real time PCR, also known as quantitative PCR or qPCR, allows for both the amplification and simultaneous quantification of targeted DNA sequences. It works by detecting amplified DNA in real time as the reaction progresses, rather than just at the end, as in standard PCR. There are two main methods for detection - using non-specific fluorescent dyes that bind to any double-stranded DNA, or using sequence-specific fluorescent probes. Real time PCR is commonly used for diagnostic applications to detect infectious diseases and cancers, as well as basic research applications to quantify gene expression levels.
Real Time PCR allows for detection and quantification of DNA as amplification occurs. It monitors fluorescence at each cycle to measure DNA accumulation. There are two main types of instrumentation - two-step qRT-PCR which involves reverse transcription followed by PCR, and one-step which combines these steps. Detection relies on fluorescent dyes like SYBR Green or target-specific Taqman probes. Real Time PCR provides advantages over conventional PCR like not requiring gels and being faster and less complex for quantification.
DNA amplification techniques include in vivo cloning and in vitro PCR. PCR was independently proposed in the 1970s and 1980s and allows selective amplification of DNA segments using a thermostable DNA polymerase. Key components of PCR include a template DNA, primers, DNA polymerase, nucleotides, and magnesium. During cycling, the DNA is denatured, primers anneal, and the polymerase extends the DNA. PCR has revolutionized molecular biology due to its ability to rapidly amplify specific DNA regions.
The document discusses biomaterials, which are materials used in medical applications that interact with biological systems. It defines biomaterials and outlines their history, characteristics, examples of applications like implants and grafts, challenges, and future potential. Key biomaterial properties include biocompatibility, mechanical compatibility with tissues, and ability to perform specific functions. Common biomaterials are metals, ceramics, polymers, and composites used in devices like heart valves, dental implants, and orthopedic implants.
PCR is a technique for amplifying DNA sequences. It requires template DNA, reaction buffer, magnesium ions, dNTPs, primers, and DNA polymerase. Variations include colony PCR, nested PCR, and real-time PCR, which uses fluorescent probes to detect amplification in real time. Common probe types are SYBR Green dyes, TaqMan probes, molecular beacons, and hybridization probes, which use FRET between donor and acceptor dyes. Real-time PCR instruments contain excitation sources and fluorometers to detect fluorescence levels during thermal cycling.
The document discusses enzyme-linked immunosorbent assay (ELISA), a technique used to detect substances like proteins. ELISA uses an enzyme to detect the binding of antibodies to antigens, producing a color change that indicates the presence of the antibody-antigen complex. There are direct and indirect ELISA methods, as well as variations like immobilized antigen ELISA for antibody detection and sandwich ELISA for antigen detection. ELISA has applications in medical diagnostics to detect antibodies in patients and test for substances like allergens in food.
A detailed description about the basic steps involved in the - PCR - Polymerase Chain Reaction, its applications,its limitations and steps to overcome it.
This document provides an overview of biomaterials used in implants. It begins with the historical background of implant materials and then classifies biomaterials according to their composition, including metals, ceramics, polymers and composites. Key properties for implant materials are discussed, including bulk properties like strength and elastic modulus, as well as surface properties like biocompatibility and corrosion resistance. Specific biomaterials are then described in more detail, including titanium, titanium alloys, cobalt-chromium alloys, and calcium phosphate ceramics. The document concludes by discussing surface characterization and preparation of biomaterials.
ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of antibodies and antigens in a liquid sample. It relies on an enzyme-linked antibody or antigen to detect the target protein. There are different types of ELISA including direct, indirect, sandwich, and competitive. The ELISA process involves coating a plate with an antigen or antibody, adding a sample and enzyme-linked antibody, washing unbound material, and detecting the enzyme's product to quantify the target. ELISAs are widely used in medical testing, food safety, and disease detection.
The ELISA (enzyme-linked immunosorbent assay) is a popular biochemistry assay that uses antibodies and color change to identify a substance. It involves using an enzyme-linked antibody to detect antigen-antibody binding, where the enzyme converts a colorless substrate into a colored product. There are several types of ELISA including indirect, direct, sandwich, and competitive ELISA. ELISA has various applications such as screening blood donations, measuring hormone levels, and detecting infections and allergens.
LncRNA WARS2-IT1 Functions as an Oncogene and is Associated with Poor Outcome...semualkaira
Glioblastoma multiforme (GBM) is one of the most common malignant brain tumors in adults and has high mortality and relapse rates. Over the past few years, great advances have been made in the diagnosis and treatment of GBM, but unfortunately, the five-year overall survival rate of GBM patients is approximately 5.1%. Our study aimed to investigate the new mechanism of Long noncoding RNAs (lncRNAs) WARS2-IT1 regulate the malignant progression of Glioblastoma.
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Glioblastoma multiforme (GBM) is one of the most common malignant brain tumors in adults and has high mortality and relapse rates. Over the past few years, great advances have been made in the diagnosis and treatment of GBM, but unfortunately, the five-year overall survival rate of GBM patients is approximately 5.1%. Our study aimed to investigate the new mechanism of Long noncoding RNAs (lncRNAs) WARS2-IT1 regulate the malignant progression of Glioblastoma.
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Glioblastoma multiforme (GBM) is one of the most common malignant brain tumors in adults and has high mortality and relapse rates. Over the past few years, great advances have been made in the diagnosis and treatment of GBM, but unfortunately, the five-year overall survival rate of GBM patients is approximately 5.1%. Our study aimed to investigate the new mechanism of Long noncoding RNAs (lncRNAs) WARS2-IT1 regulate the malignant progression of Glioblastoma.
This document describes a method for performing RNA sequencing on single nuclei. Key points:
1) The authors demonstrate that double-stranded cDNA can be synthesized from single mouse neural progenitor cell nuclei and hippocampal tissue nuclei, allowing for whole transcriptome sequencing.
2) On average, sequencing of single nuclei detected over 16,000 of the approximately 24,000 mouse protein-coding genes.
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Bright is a transcription factor required for both hematopoietic stem cell development and B cell lineage development. Mice lacking Bright die by mid-gestation due to failed hematopoiesis. Rare Bright-deficient mice that survive have deficits in hematopoietic stem cells and impaired B cell development and function, including reduced natural antibody and IgG1 class switching responses. Bright is thus necessary for both the formation of hematopoietic stem cells and their differentiation into specific blood lineages such as B cells.
Effect of stemregenin1 and sb431542 small molecules on ex vivo expansion of u...Liberty University (LU)
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This document discusses the role of the guanine nucleotide exchange factor C3G in neuronal differentiation. It finds that C3G protein levels increase when human neuroblastoma cells are induced to differentiate through serum starvation or treatment with forskolin or nerve growth factor. Overexpression of C3G stimulates neurite growth and increases responsiveness to differentiation signals, in a process dependent on C3G's catalytic domain and the functions of Rap1 and Cdc42. Knockdown of C3G inhibits forskolin- and nerve growth factor-induced differentiation and enhances cell death from serum starvation. C3G phosphorylation and localization to the Golgi are increased by forskolin and nerve growth factor treatment, and C3G
1. The document discusses lateral root development in Arabidopsis thaliana, including the initiation and stages of lateral root primordia formation. Key processes like cell division and emergence are described.
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2011 - Cellular inhibitor of apoptosis protein-1 (cIAP1) can regulate E2F1 tr...Simon Gemble
Cellular inhibitor of apoptosis protein-1 (cIAP1) can directly interact with the transcription factor E2F1 and increase its transcriptional activity. cIAP1 is recruited to E2F1 binding sites on cyclin E and cyclin A promoters in a cell cycle-dependent manner. Silencing cIAP1 inhibits E2F1 DNA binding and transcriptional activation of cyclin E, reducing cell proliferation. Thus, one function of nuclear cIAP1 is to regulate E2F1 transcriptional activity and control cell cycle progression.
1) The C-terminal carboxylate group of integrin β1 is necessary for its association with the kindlin-2 adapter protein. Affinity measurements indicate this interaction is coordinated by a putative carboxylate-binding motif in the FERM subdomain F3 of kindlin-2.
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This study found that oncogenic Ras sensitizes normal human cells to TRAIL-induced apoptosis by enhancing caspase 8 recruitment and activation at the DISC. Specifically:
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This document summarizes research investigating the role of the transcription factor Irx3 in regulating angiogenesis. The key findings are:
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So in summary, this research identifies Irx
Cloning and sequence analysis of banana streak virus dna. harper 1998Paloma Susan
Banana streak virus (BSV) causes severe problems for banana cultivation. The researchers cloned and sequenced the genome of a Nigerian isolate of BSV. The genome was found to be 7,389 base pairs and organized similarly to other badnaviruses, with three open reading frames. Comparison showed BSV is distinct from but closely related to sugarcane bacilliform virus. PCR primers designed from the sequence data detected BSV sequences in banana plants, indicating portions of the BSV genome may integrate into the banana genome. The BSV sequence provides a basis for more sensitive PCR-based detection methods.
The document describes a new reporter system for evaluating the specificity and efficacy of CRISPR/Cas9 and guide RNAs (gRNAs). The system uses an EGFP reporter plasmid containing the target sequence of interest. When co-transfected with Cas9 and the corresponding gRNA, double strand breaks at the target site disrupt EGFP expression. This allows unbiased measurement of Cas9 and gRNA activity levels. The document demonstrates that mismatches between gRNAs and target sequences can occur anywhere in the gRNA and depend on both the gRNA sequence and Cas9 construct used. This reporter system promises to improve genomic engineering success rates by facilitating selection of optimal gRNA and Cas9 combinations.
Isolation of an adult blood derived progenitor cell population capable of dif...lifextechnologies
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This document summarizes a study that used cDNA-amplified fragment length polymorphism (AFLP) analysis to identify approximately 1,340 plant genes whose expression levels change periodically during the cell cycle. Tobacco Bright Yellow-2 cells were synchronized and sampled at various time points to analyze genome-wide expression patterns. Hierarchical and quality-based clustering grouped the periodically expressed genes into clusters corresponding to different cell cycle phases like S, G2, and M phases. Many of the identified genes have unknown functions or no known homology, indicating many unknown processes underlying cell division in plants. Core cell cycle regulatory genes like cyclins showed expression profiles matching their known roles. The study provides new insights into plant-specific cell cycle regulation and cell
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2. zebrafish Flk1 does not phenocopy the murine Flk1 knockout, we
searched the zebrafish genome for other VegfA genes and FLK1/
KDR homologues.37 By comparison of consensus mammalian
VEGFA and KDR sequences to the available zebrafish genome, we
indeed identified a duplicated vegfA gene (hereafter referred to as
vegfAb) and a putative KDR gene (hereafter referred to as kdrb).
Zebrafish embryos with reduced levels of VegfAb, and sepa-
rately Kdra and Kdrb, show defects in vascular development. In
addition, in vitro overexpression of either the 171- or 210-aa
isoforms of VegfAb in tissue culture cells demonstrates that both
VegfAb isoforms are poorly secreted, whereas both VegfAa121 and
VegfAa165 isoforms are highly secreted. However, both VegfAa and
VegfAb can bind Kdra and Kdrb in vitro. These data support a role
for multiple VegfA and type III RTKs in zebrafish vascular
development.
Materials and methods
Zebrafish husbandry
Zebrafish were maintained and staged as described.38
Cloning, sequencing, RT-PCR, and RACE reactions
Using versions 2 and 3 of the Sanger Center zebrafish genome assembly, we
used a standard tblastn algorithm to find the duplicated genes. We designed
primers based on search results and isolated the full-length cDNA clones
using a combination of oligo dT reverse-transcription–polymerase chain
reaction (RT-PCR; BD Clontech, Palo Alto, CA) and rapid amplification of
cDNA ends (RACE; BD Clontech). We isolated total RNA for the RT-PCR
and RACE reactions from the TU strain using Absolutely RNA (Stratagene,
La Jolla, CA) and used it for the RT-PCR and RACE reactions. RT-PCR of
the open reading frames required amplification by nested PCR to visualize a
product by agarose electrophoresis. Appropriately staged cDNA was first
amplified in an MJ thermocycler (MJ Research, Watertown, MA) for
40 cycles (denaturing: 94°C for 20 seconds; annealing: 58°C for 30 seconds; and
extension: 72°C for 1 minute) using the 5Ј primer CCCTGTGTGAAGAAGC-
CGCTACT and 3Ј primer CAACCACTTCACTTCATTCTCTCG. The result-
ant reaction was diluted 1/100 and 2 L used as template for a subsequent PCR
reaction using the 5Ј primer GCTTTCTGAGACCTTTACCAATGC and
3Ј primer TCTCAAGTCAGTTCGGTTCACCTCC for 30 more cycles. The
GenBank accession numbers are DQ026828 for vegfAb210, DQ150576 for
vegfAb171, and DQ026829 for kdrb.
In situ hybridization and photography
Whole-mount in situ hybridizations were performed as previously de-
scribed.31,39 Antisense RNA probes corresponded to the complete ORF of
each of the genes. Processed embryos were photographed in 90% glycerol
on a Leica MZFLIII dissecting microscope (Leica Mikrosysteme Vertrieb,
Wetzlar, Germany), PLAN Apo 1.0 objective, 8ϫ-100ϫ magnification.
Images were acquired using a Zeiss Axiocam MNRC5 camera and
Axiovision software version 4.001(both from Carl Zeiss, Thornwood, NY).
Alkaline phosphatase and hemoglobin staining
O-dianisidine staining for hemoglobin was performed as previously de-
scribed.31 Alkaline phosphate staining of the vasculature was carried out by
4% paraformaldehyde fixation of the embryos for 2 hours at room
temperature, followed by immersion in acetone for 30 minutes at Ϫ20°C.
The embryos were rinsed twice in 1 ϫ PBST (Gibco-BRL, Carlsbad, CA)
and equilibrated in staining buffer (NTMT, 100 mM Tris pH 9.5, 50 mM
MgCl2, 100 mM NaCl, 0.1% Tween-20) 3 times for 15 minutes each. The
vasculature was visualized by staining in NTMT containing NBT and BCIP
for 5 to 20 minutes.
Morpholino injections
A 1-nL volume of a 150-M to 2-mM morpholino (Gene-Tools, Philomath,
OR) dissolved in 1 ϫ Danio buffer containing 100 M of a fluorescein
labeled control morpholino was injected into either AB or transgenic fli-gfp
embryos at the 1- to 4-cell stage.
The sequences of the injected morpholinos were as follows: VegfAa:
GTATCAAATAAACAACCAAGTTCAT; VegfAb ATG: GGAGCACGC-
GAACAGCAAAGTTCAT; VegfAb-75: TCCGATGATAGGCGCACTT-
TAGCAA; Kdra: CCGAATGATACTCCGTATGTCACTT; Kdrb-m1: TAT-
GCTCTATTAGATGCCTGTTTAA; and Kdrb-m2: CAGTTATGCT-
CTATTAGATGCCTGT.
Rescue of the VegfAb morpholino VegfAb-75 was accomplished by
subcloning the complete open reading frames (ORFs) of both Vegfab
isoforms in PCS2ϩ, and production of capped mRNA of each isoform
separately (Ambion, Austin, TX). RNA (50 pg of each) was then simulta-
neously injected with the morpholino. All injected embryos were photo-
graphed live on a Zeiss Axiovert (fluorescence) using a Zeiss Axiocam and
Axiovision software.
Affymetrix microarrays
Embryos injected with either the VegfAa, VegfAb-75 morpholinos, or
controls were grown in standard embryo medium until 24 hours after
fertilization (hpf). Embryos were then disaggregated in Trizol. Isolation of
cDNA, hybridization, and analysis of the microarrays were accomplished
as previously described.40
Cell culture and Western blotting
Chinese hamster ovary (CHO) cells, African green monkey kidney cells
(Cos7), and human embryonic kidney cells (HEK293) were purchased from
American Type Culture Collection (ATCC, Manassas, VA). Cell culture and
transfection were performed in principal as previously described.41 After
48 hours of incubation, conditioned medium of transfected cells was
collected and the transfected cells were lysed with lysis buffer (50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate,
0.1% SDS,
1 mM EDTA) supplemented with protease inhibitor cocktail (Complete
Mini; Roche, Indianapolis, IN). The conditioned medium and total cell
lysates were resolved by 6% and 10% sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) for Flk1 and VegfA,
respectively. For detection of Flk1 or VegfA proteins, the blots were
incubated with mouse monoclonal anti-V5 antibody (1:5000 dilution;
Invitrogen, Frederick, MD)
Ligand-receptor binding
HEK 293 cells were transfected with Ig-HA-vegfAa165-myc/pSecTag2,
Ig-HA-vegfAb171-myc/pSecTag2, soluble kdra-V5/pcDNA3.1, and soluble
kdrb-V5/pcDNA3.1. After 4 days of incubation, the conditioned media
were collected. The amount of VegfA and RTK was estimated by Western
blotting with anti-Myc antibody (1:2000 dilution) and mouse monoclonal
anti-V5 antibody, respectively. The conditioned media were mixed and
incubated at 4°C overnight with agitation. VegfAproteins were pulled down
by antirabbit polyclonal V5 antibody (Novus Biologicals, Littleton, CO)
and the complexes resolved by 6% SDS-PAGE. For detection of VegfA
bound to Flk1, the membranes were incubated with mouse monoclonal
anti-Myc antibody.
Results
Cloning and analysis of VegfAb and Flk1
The zebrafish vegfA gene located on zebrafish chromosome 16 encodes
multiple isoforms, but no apparent homologues of human VEGF145,
VEGFA189, or VEGFA206. We postulated that a second, duplicate
zebrafish VegfA gene might encode the missing isoforms. Using a
3628 BAHARY et al BLOOD, 15 NOVEMBER 2007 ⅐ VOLUME 110, NUMBER 10
For personal use only.on October 24, 2014.by guestwww.bloodjournal.orgFrom
3. standard tblastn algorithm, short sequence segments with homology to
mammalian VEGFA and KDR were identified using the zebrafish
genome assembly versions 2 and 3, available at the Sanger Center
website(http://www.sanger.ac.uk/Projects/D rerio/).Wefoundadupli-
cate vegfA gene, hereafter referred to as vegfAb to distinguish it from the
previously cloned vegfA gene (now termed vegfAa). This DNA se-
quence then served as the starting point for isolating the complete cDNA
from TU fish using a combination of RACE and RT-PCR. Radiation
hybrid mapping of this gene placed vegfAb on zebrafish chromosome 4,
whereas vegfAa is located on chromosome 16 (Yi Zhou, personal
written communication), November 2003).
The full-length vegfAb cDNA contained 2814 nucleotides
(nt’s) and a predicted ORF of 233 aa’s and encoded a putative
protein with stronger homology to VEGFA than VEGFB,
VEGFC, VEGFD, or PLGF (Figure 1). Therefore, the newly
identified gene appeared to be a duplicated zebrafish vegfA gene.
By RT-PCR, expression of 2 differentially spliced mRNAs was
detected from the early embryo (8 hours after fertilization [hpf])
through 4 days after fertilization (dpf) (data not shown). The
larger of 2 bands corresponded to an isoform of 210 aa’s
(VegfAb210), the smaller to a second isoform of 171 amino acids
(VegfAb171). The 2 zebrafish VegfA proteins (VegfAa and
VegfAb) were identical in size and exon structure to human
VEGFA through the first 138 aa’s (exons 1-5), suggesting
common ancestry (Figure 2A); however, insertions and dele-
tions occurred in the C-terminus, primarily in exons
6 and 7. Analysis of the genomic structure of the vegfA isoforms
by blasting against the available zebrafish genome showed 6
exons for vegfAa121, 7 for vegfAa165 and vegfAb171, and 8 for
vegfAb210; vegfAb171 and vegfAb210 share exons 1 to 5, 6, and 7
(Figure 2A). Figure 2 shows that exon use is highly conserved
between humans and zebrafish, but also that exon 6a, which
promotes mammalian VEGF binding to the basement membrane
in VEGFA145, VEGFA189, and VEGFA206, is found only in
vegfAb210. We suggest that zebrafish vegfAb210 may be the
functional equivalent of these particular VEGFA isoforms and
that vegfAb171 is orthologous to mammalian VegfA165.
By similar methods, we also obtained a 5360-nt kdrb cDNA that
encoded a novel zebrafish KDR orthologue. Alignment of critical
tyrosine residues between the zebrafish Kdrb cytoplasmic domain
and human and mouse KDR (VEGFR2) showed that the flk1-
related RTK described here is more closely related to KDR than the
previously described flk1 gene.31 In humans, tyrosine residue
Y1175 has been reported as a binding site for PLC␥1 and
Src-homology 2 protein (Shb) in -cells.42 The corresponding
tyrosine residue in the mouse, Y1173, has recently been identified
as an essential tyrosine residue for vasculogenesis and hematopoi-
esis, as knock-in mice substituting it with phenylalanine exhibited
severe vascular and hematopoietic defects, similar to KDR-null
mice.43 This critical tyrosine residue was conserved in both
zebrafish Flk1 genes (Figure 2B). However, 4 important tyrosine
residues in the C-terminal tail of KDR were conserved only in the
zebrafish RTK gene reported here (Kdrb), but not in the previously
identified RTK (Figure 2B). These residues, Y1214, Y1305,
Y1309, and Y1319, mediate binding of focal adhesion kinase
(FAK), which in turn interacts with PI3 kinase and paxillin.44-47 By
radiation hybrid mapping (Yi Zhou, personal communication), the
newly identified KDR gene was located on chromosome 20 near
the zebrafish orthologues of KIT and PDGFRA, thereby establish-
ing synteny between the zebrafish, human, and mouse KDR
chromosomal regions (Figure 1C). Based on structural conserva-
tion, synteny, and biochemical data, we suggest that this RTK gene
(Kdrb) is a duplicate of the previously cloned gene flka, which we
suggest should be called Kdra. Another duplicated zebrafish type
III RTK with homology to human FLT1 (Figure 1) is located on
zebrafish chromosome 24, near the zebrafish orthologue of IPF and
CDK8 (Yi Zhou, personal communication). Although this gene is
not the subject of this paper, we suggest that it be called flt1
henceforth. The cloning and function of this FLT1 gene will be
Figure 1. Evolutionary relationship between human
and zebrafish Vegfa isoforms and receptors. Protein
sequences were aligned using ClustalW (European Bioin-
formatics Institute, Cambridge, United Kingdom) with the
complete open reading frames. Species are indicated
as follows: zf, zebrafish; m, mouse; and h, human.
(A) Alignment of VegfA proteins shows a closer relation-
ship of the newly isolated gene to VEGFA than VEGFB,
VEGFC, VEGFD, and PLGF. (B) Alignment of RTK
sequences. The zebrafish gene (Kdra) formerly referred
to as flk1 shows a closer relationship to hFLT1 and mFlt1
than to hKDR (hFLK1) and mKDR (mFlk1). (C) Radiation
hybrid mapping of zebrafish kdra, flt1, and kdrb and
synteny comparison. Zebrafish kdrb is located on chromo-
some 20 in a region syntenic to human KDR, nearby the
zebrafish orthologues of kit and pdgfra, which map near
human KDR on human chromosome 4q12. Zebrafish
kdra is on chromosome 14, and flt1 near cdk8 and ipf1 in
a region on chromosome 24, which is syntenic to the
human 13q12 region where FLT1, CDK8, and IPF1 are
located.
DUPLICATED VEGF AND KDR GENES IN ZEBRAFISH 3629BLOOD, 15 NOVEMBER 2007 ⅐ VOLUME 110, NUMBER 10
For personal use only.on October 24, 2014.by guestwww.bloodjournal.orgFrom
4. described elsewhere (Stefan Schulte-Merker, personal written
communication, February 2005).
Expression of vegfAb and kdrb during embryogenesis
To analyze the temporal and spatial expression of the zebrafish
vegfAb gene, we used whole-mount in situ hybridizations of
wild-type embryos (Figure 3A). Both vegfAa isoforms were
diffusely expressed at 12 somites, whereas at 24 hpf, although still
broadly expressed, vegfAa165 was more evident in the somites
compared with vegfAa121. At 2 dpf, expression of vegfAa165 was
nearly absent, whereas vegfAa121 is still expressed in the develop-
ing heart and associated vasculature, head, and around the develop-
ing eye. There was no clear expression of vegfAa165 at 4 dpf, and
vegfAa121 was restricted to developing pharyngeal structures and a
subset of the developing CNS. A probe detecting both isoforms of
vegfAb demonstrated significantly less expression at 12 somites
compared with either vegfAa isoform. At 24 hpf, there was
significant expression throughout the CNS and detectable, although
significantly less than seen with vegfAa165, expression within the
developing somites. At 2 dpf, expression was greatest surrounding
the developing eye and retina, the pharyngeal arches, and glo-
meruli. At 4 dpf, expression persisted in vasculature surrounding
the eye, and the glomeruli.
The expression of kdrb was indistinguishable from the expres-
sion of kdra (Figure 3B). Kdra was first expressed at about 7 to
8 somites in the bilateral stripes that contain the developing
angioblasts (data not shown) and then localized to the intermediate
cell mass (ICM) and the developing vasculature. By 30 hpf, kdra
was expressed in the major trunk, head, and intersomitic vessels
and persisted through 4 dpf. The expression patterns of kdra and
kdrb in the major vessels are consistent with roles for both
receptors in vascular development.
VegfAb morphants show defects in angiogenesis
To determine whether VegfAb is required for vasculogenesis,
angiogenesis, and hematopoiesis, either wild-type AB or transgenic
fli1-gfp embryos were injected with morpholinos against vegfAb,
and development of their vasculature and hematopoiesis was
assayed. Injection of a vegfAb morpholino either against the start
codon or the 5Ј untranslated region (UTR) caused defects in
angiogenesis evident at 2 dpf (Figure 4A), with extravasation of
blood into the surrounding tissue beginning at about 3 to 4 dpf
(Figure 4E,F,J), but had no effect on vasculogenesis, as judged by
direct fluorescent visualization and alkaline phosphatase staining
(data not shown). Indeed, red blood cells (RBCs) circulated
unimpeded through the axial vasculature. The regular expression
patterns of markers of early hematopoiesis in injected embryos,
gata1 at 15 hpf and E3 globin at 30 hpf (data not shown), suggest
blood development is normal in VegfAb morphants.48
Beginning at 3 dpf, the number of circulating RBCs gradually
decreased in 63% (162/255) of the embryos injected with 4.5 ng of
the vegfAb start codon morpholino and in 47% (74/156) with 9 ng
of the vegfAb 5ЈUTR morpholino. At the same time, RBCs were
directly seen accumulating in the perivascular tissues, especially in
the brain and in the eyes (Figure 4J), accounting for the drop in
circulating RBCs. This loss was accompanied by increasing
pericardial edema and death of the injected embryos between 5 to
7 dpf. Beginning at 2 dpf, injected embryos also showed defects in
angiogenesis, particularly in the formation of intersegmental
vessels and subintestinal veins (Figure 4B,G). The subintestinal
Figure 2. Expression and structure of zebrafish VegfA genes
and receptors. (A) Zebrafish vegfA exon/intron boundaries were
predicted by blasting against the zebrafish genome. The structural
similarities between vegfAb171, vegfAa165, and human VEGFA165
suggest that vegfAb171 is an orthologue of human VEGFA165 and
similarly, that vegfAb210 is orthologous to human VEGFA189 and/or
VEGFA206. (B) Alignment of the cytoplasmic domain of zebrafish
Flk1 and human and mouse KDR. Conservation of critical tyrosine
residues (arrows) shows that the flk1 RTK described here (Kdrb)
is more closely related to KDR than the previously described
putative flk1 gene (referred to now as kdra). See “Cloning and
analysis of VegfAb and Flk1” for a full description of the critical
residues and their function.
3630 BAHARY et al BLOOD, 15 NOVEMBER 2007 ⅐ VOLUME 110, NUMBER 10
For personal use only.on October 24, 2014.by guestwww.bloodjournal.orgFrom
5. vein defects ranged from near total absence to an erratically placed
and thin vasculature (Figure 4I), the intersegmental vessels often
missing or prematurely terminated (Figure 4B,D). These defects
were more pronounced anteriorly, especially near the developing
head and perhaps accounting for the tendency for blood accumula-
tion in this area.
The VegfAa morphant phenotype differs significantly from that
of VegfAb morphants. Whereas VegfAb morphants have normal
vasculature and hematopoiesis until 2 dpf, VegfAa morphants have
a nearly complete absence of the major intersegmental vessels at
2 dpf and reduced hematopoiesis (data not shown and Nasevicius et
al22). Because injecting morpholinos against vegfAb should not
alter the VegfAa121 and VegfAa165 isoforms, we wanted to test if
coinjection of morpholinos against vegfAa and vegfAb might result
in a more severe phenotype than that seen in VegfAa morphants.
However, coinjection of morpholinos against vegfAa and vegfAb
caused a vascular phenotype indistinguishable from that of VegfAa
morphants (data not shown) and identical to the phenotype
previously described for VegfAa morphants.20,22 These data suggest
that that the role of VegfAa in angiogenesis is distinct from that of
VegfAb.
Kdrb acts synergistically with Kdra
Targeted deletion of the Flk1 gene in the mouse leads to death
around E9.5 with severe defects in both vasculogenesis and
hematopoiesis.6 Because developmental pathways are often con-
served between mice and zebrafish, we sought to determine
whether the zebrafish Kdrb receptor, alone or in combination with
Kdra, might also play a role in vasculogenesis or hematopoiesis.
Embryos were first injected with 2 independent morpholinos
directed against the 5Ј UTR of kdrb. Injection of 4.5 ng of either
morpholino did not lead to any demonstrable defect in angiogen-
esis. Variable defects in angiogenesis seen in 45 (40%) of
112 embryos injected at higher doses (Ͼ 9 ng/embryo) were also
visualized in approximately 8% of an equal amount of 5-bp
mismatch control morphants (14/184), suggesting this might due to
nonspecific toxicity of the morpholinos. Hematopoiesis was unaf-
fected in Kdrb morphants, as shown by whole-mount in situ
hybridization with gata1 at 18 hpf and with E3 globin at 30 hpf
(data not shown).
Coinjecting kdrb and kdra morpholinos resulted in embryos in
variable defects in the formation of the posterior vasculature, and
most dramatically the intersegmental arteries (Figure 5C,F,I).
These coinjection experiments suggested that Kdra and Kdrb
cooperate in the growth, proliferation, and remodeling of the
developing zebrafish embryonic vasculature, and particularly for-
mation of the arteries. Compared with the murine Flk1 knockout
phenotype, however, the phenotype of zebrafish coinjected with
kdra and kdrb morpholinos was less severe and did not appear to
affect hematopoiesis. Because of the large numbers of receptors
mediating the VegfA signal and technical limitations on how many
can be knocked down by morpholino injections, it is currently not
possible to analyze vasculogenesis and angiogenesis in embryos
lacking all VegfA activity. We therefore cannot determine whether
the weaker phenotype resulted from residual Kdr-like activity or
from species-dependent differences in receptor function.
Microarray analysis of VegfAa and VegfAb morphants
Although the expression patterns and morpholino analysis of
vegfAa and vegfAb suggest that the 2 genes may function through
different pathways, the magnitude of the differences is unclear. To
help compare and contrast the pathways affected by the 2 genes, we
determined the transcriptional profile of the 2 morphants by gene
expression analysis. Affymetrix zebrafish GeneChips, containing
approximately 14 900 zebrafish transcripts were used to examine
the transcript expression of vegfAa and separately vegfAb mor-
phants at 24 hpf, when the vegfAa morpholino phenotype is clearly
evident and the embryos viable, and vegfAb was expressed. Two
individual injections of vegfAb and vegfAa morphants compared
with injected morpholino controls were made.
There was excellent correlation between the 2 injections of each
morpholino (data not shown), and no obvious nonspecific toxicities
were noted. We based further analyses on 302 genes in vegfAa
morphants, 301 genes in vegfAb morphants, and 39 genes common
to both morphants that were significantly regulated (Tables S1-S3,
available on the Blood website; see the Supplemental Materials
link at the top of the online article). When available through Zfin
(http://zfin.org/cgi-bin/webdriver?MIvalϭaa-xpatselect.apg), the
whole-mount in situ expression of the regulated gene is noted.
Figure 3. Expression of the zebrafish VegfA and Kdra genes in the developing
embryo. (A) Whole-mount in situ hybridization with probes to vegfAa121, vegfAa165,
or vegfAb. Both vegfAa121 and vegfAa165 are diffusely expressed at 12 somites,
whereas vegfAb is not appreciably expressed at this time point. At 24 hours after
fertilization (hpf), both isoforms of vegfAa were broadly expressed, although veg-
fAa165 was more clearly seen in the somites. VegfAa165 is more highly expressed in
the developing CNS than vegfAb, whereas in the lens vegfAb is more highly
expressed, and both are expressed in the developing somites (˜). At 2 dpf, vegfAa121
is expressed in the developing heart vasculature and pectoral fins. The aortic
vasculature is also identified in vegfAb in situs, but in contrast, only vegfAb is
expressed in the developing pronephros (Š). At 4 dpf, significant expression is
restricted to vegfAb in the vasculature surrounding the eye. (B) Wild-type embryos
were analyzed by whole-mount in situ hybridization with a probe to either kdrb (top
panels) or kdra (bottom panels) at 16 somites (left panels), 2 dpf (middle panels), and
4 dpf (right panels). At 16 somites, expression is limited to the inner cell mass. By 2
dpf, the developing intersomitic vasculature expresses both kdrb and kdra. At 4 dpf,
both genes are expressed in the developing subintestinal veins (SIVs) and in the
remaining vasculature. See “In situ hybridization and photography” for image
acquisition information.
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6. A large number of genes affected in the vegfAa morphant that had a
specific expression pattern were found to be expressed in the
developing somites, myotomes, and CNS, while similarly, a
number of genes expressed in the pharyngeal arches, CNS, eye, and
lens were noted in vegfAb morphants. No clear expression pattern
consensus emerged from the analyses of the commonly regulated
vegfAa and vegfAb morphant genes.
We subsequently mapped our zebrafish expression values to
human genes and then uploaded the mapped expression values for
analysis with Ingenuity’s Pathway Analysis program (IPA; Ingenu-
ity Systems, Redwood City, CA), a human curated database of gene
interactions. For the vegfAa morphant, the largest numbers of genes
(37) have been implicated in “small molecule biochemistry,”
followed by 30 in “skeletal and muscular system development and
function.” In contrast, in vegfAb morphants the largest number of
genes (71) are implicated in “cellular growth and proliferation.”
A similar analysis of the 39 genes commonly altered in both vegfAa
and vegfAb morphants did not clearly reveal particular pathway
associations (data not shown).
Secretion and binding of VegfA and Flk1
VegfAa and vegfAb constructs encoding the entire ORF and fused to
a 3Ј terminal V5 tag were independently transfected into Chinese
hamster ovary (CHO), COS7 (monkey kidney), and PAE (porcine
aortic endothelial) cell lines (Figure 6). Although all 4 VegfA
isoforms could be detected in cell lysates using a V5 antibody
(Figure 6A), neither VegfAb171 nor VegfAb210 were secreted into
the conditioned medium (Figure 6B), despite a well-defined signal
peptide. Both Kdra and Kdrb can be detected in PAE cell lysates
after transfection (Figure 6C).
To determine the ligand-receptor specificity, we analyzed the
binding of soluble receptors tagged with V5 to VegfA isoforms
tagged with Myc using the Ig signal peptide in place of the
VegfAb signal peptide, by which secretion of VegfAb in mamma-
lian cells occurs (Figure 6D). HEK 293 cells were separately
transfected with Ig-HA-vegfAa165-Myc, Ig-HA-vegfAb171-Myc,
soluble kdra-V5, and soluble kdrb-V5. The conditioned VegfA and
sRTK medias were pairwise mixed, incubated overnight, and then
immunoprecipitated with anti-V5 antibody. Bound protein was
detected by Western blot using an anti-Myc antibody (data not
shown). Soluble Kdra bound to both VegfAa165 and VegfAb171;
however, Kdrb did not appear to bind VegfAb171 as well as
VegfAa165 in this assay. As human VegfA has been shown to bind
multiple RTKs, we wanted to analyze if the different zebrafish
VegfA genes and isoforms acted similarly.49 The zebrafish VegfA
isoforms with an Ig signal peptide and both HA and V5 tags were
transfected into COS7 cells, and their expression was confirmed by
blotting with an anti-V5 antibody (Figure 6F). Kdra and kdrb were
Figure 4. Vascular defects in VegfAb morphants.
(A) Photomicrograph of a 5-bp mismatch vegfAb morpho-
lino–injected (4.5 ng) fli1-gfp embryo at 2 dpf demon-
strates no vascular defects (only one shown, although no
phenotype was seen with either). (B) Defects in the
formation of the ISVs anteriorly are clearly visible begin-
ning at 2 dpf. (C) Photomicrograph of a 5-bp mismatch
vegfAb control morpholino–injected (4.5 ng) fli1-gfp em-
bryo at 4 dpf for comparison. (D-F) Beginning at 3 dpf, the
number of circulating RBCs gradually decreased in 63%
(162/255) of the embryos injected with 4.5 ng of the
vegfAb ATG morpholino and in 47% (74/156) with 9 ng of
the vegfAb-75 (5ЈUTR) morpholino (day-4 embryos are
shown). Injection of an equivalent amount of 5-bp mis-
match morpholinos did not affect vasculogenesis, and
coinjection of the active vegfAb-75 (5ЈUTR) morpholino
together with 50 pg each of vegfAb171 and vegfAb210 RNA
reduced the number of embryos with defects in vasculo-
genesis by more than 50% (8/32 vs 22/40 abnormal
embryo). VegfAb morphants showed decreased interseg-
mental vessel number and size (˜, Š in panels D and F)
and aberrant head vascular development. SIVs were
severely reduced in number, size, and branching that was
more pronounced anteriorly. (E) At 4 dpf, blood is appar-
ent in the head and anterior embryos (˜), which
(F) corresponds to the areas with defects in angiogen-
esis. (G) Control morpholino–injected embryos demon-
strate normal subintestinal vein (SIV) architecture by
alkaline phosphatase staining, and (H) RBCs are shown
by staining with o-dianisidine that stains hemoglobin
reddish/brown. However, (I) injection of either morpholino
targeting VegfAb leads to SIVs that are erratically placed
and thin or nearly completely absent (only the start codon
morpholino is shown) and (J) extravasation of RBCs in
various structures, which is more pronounced anteriorly
where the angiogenic defects are most visible (B-F). The
results are combined from at least 3 separate experi-
ments, and the photomicrographs are representative of
the visible defects.
3632 BAHARY et al BLOOD, 15 NOVEMBER 2007 ⅐ VOLUME 110, NUMBER 10
For personal use only.on October 24, 2014.by guestwww.bloodjournal.orgFrom
7. separately transfected into these same cell lines with and without
vegfAa165 or vegfAb171. Both vegfAa165 and vegfAb171 were able to
activate both RTKs as demonstrated by antiphosphotyrosine anti-
body staining (Figure 6E). Therefore, the zebrafish VegfAa and
VegfAb isoforms can both bind and activate Kdra and Kdrb.
Discussion
We report here the cloning and characterization of duplicated
zebrafish genes that regulate vasculogenesis and angiogenesis. In
addition to the reported VegfA locus, now renamed vegfAa, we find
a second locus, vegfAb, which contains homologues of some of the
mammalian VegfA isoforms not encoded by vegfAa. We also
identified a second zebrafish RTK (Kdrb) with homology to
mammalian KDR.
Expression of VegfAa and VegfAb during embryonic
development
Whole-mount in situ hybridization of vegfAa and vegfAb revealed
differing expression patterns of these 2 genes (Figure 3A). Whereas
both vegfAa splice variants were broadly expressed at 12 somites,
by 24 hpf, vegfAa165 was expressed in the CNS and the somites,
whereas VegfAa121 RNA was more broadly distributed, although at
48 hpf it was expressed within/near the developing heart and
vasculature. These data differ from the reported expression of
VegfA during zebrafish embryogenesis. In that study, an unspecified
probe that detected both splice variants was similarly seen in the
prospective cardiac vasculature and somites but also in the
pronephros (seen in the present studies with vegfAb165) and was not
seen as diffusely within the CNS. The reasons for these differences
are unclear but might be related to the differing time points
analyzed and the probe chosen for use in these analyses. In the
present studies, we separately used the ORF of each isoform,
whereas the previous studies were done with an unspecified probe
that recognized both alleles, and that perhaps included 3ЈUTR
sequences as well. Our whole-mount in situ hybridization data also
correlated with our microarray data. The expression of genes
whose known embryonic whole-mount in situ pattern is within the
developing CNS and myotomes was more likely to be altered in
vegfAa morphants, whereas expression of genes in the CNS, eyes,
lens, and pharyngeal arches was altered in vegfAb morphants.
Interestingly, Hiratsuka et al (2005) examined the localization of
VegfA by immunohistochemistry during murine development and
found expression throughout the developing embryo, but especially
in the anterior part of the embryo.50 This pattern more closely, but
not completely, resembles the cumulative expression of both
zebrafish VegfA genes than any one gene or splice variant. These
expression patterns suggest that the various functions of mamma-
lian VEGFA may be encompassed within more than just these
2 zebrafish VegfA genes.
The role of zebrafish Vegf and Kdr in vascular development
In VegfAb morphants, early vascular and hematopoietic development
appeared normal at first, however as early as 2 dpf, morphants had
defects in angiogenesis. Concomitant with the vascular phenotype,
blood began to extravasate into the surrounding tissues beginning at 3
dpf (Figure 4). This extravasation of blood into the tissues surrounding
the eye and CNS in vegfAb morphants is consistent with the later
expression of vegfAb RNA in the vasculature surrounding the eye
(Figure 3A), and CNS. Similar defects in angiogenesis are seen in Kdra
morphants and the previously described zebrafish Kdra mutant.37 The
intersomitic vascular defects seen in Kdra morphants appeared to
encompass more of the embryo (Figure 5H) than those in either VegfAb
Figure 5. Kdra and Kdrb cooperate to mediate vasculogenesis during zebrafish embryogenesis. Fli-gfp transgenic embryos were injected with combinations of
morpholinos against kdra and kdrb or 5-bp mismatch control morpholinos. (A) Twenty-eight hpf embryos injected with either of 2 5-bp mismatch control morpholinos (4.5 ng
each) corresponding to separate 5ЈUTR kdrb morpholinos (m1-kdrb [n ϭ 32] and m2-kdrb [n ϭ 44]) or the previously published kdra morpholino (m-kdra [n ϭ 38]) had no
vascular abnormalities. (B) Injection of 4.5 ng kdra or kdrb morpholino has no demonstrable effect on vasculogenesis at 28 hpf (107 embryos). Vascular defects were seen at
higher concentrations of kdrb morpholinos; however, similar defects, but at a reduced penetrance, were also seen with equivalent amounts of the mismatched controls,
suggesting these effects were not directly related to Kdrb function. (C) Coinjection of either 4.5 ng m1-kdrb or m2-kdrb with 4.5ng kdra morpholino caused variable loss of
intersegmental arteries in 42 (24%) of 178 kdra/m1-kdrb or 53 (38%) of 138 kdra/m2-kdrb morpholino–injected embryos. (D) RBCs are seen in the anterior axial vasculature but
cannot circulate. (E,F) Kdra/kdrb double morphant defects are still seen at 2 dpf. (G-I) At 4 dpf, injection of 4.5 ng kdra morpholino led to defects in the angiogenesis (Š, ˜) in
34 (33%) of 101 injected embryos. Embryos coinjected with morpholinos targeting both kdra/b had severe axial vessel defects, although some subintestinal vasculature is
apparent. The results are combined from at least 4 separate experiments and the photomicrographs are representative of the visible defects.
DUPLICATED VEGF AND KDR GENES IN ZEBRAFISH 3633BLOOD, 15 NOVEMBER 2007 ⅐ VOLUME 110, NUMBER 10
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8. morphant, whose defects were more pronounced anteriorly (Figure
4B,D). Whether this result is secondary to redundant signals activating
Kdra or variable penetration of the VegfAb and Kdra morpholino
phenotype is uncertain. Reducing all known VegfA signals by coinjec-
tion of morpholinos against vegfAa (previously described in Nasevicius
et al22) and vegfAb resulted in the same vascular phenotype seen in
VegfAa morphants. Loss of VegAa and VegfAb signaling in the
zebrafish does not cause death in the developing embryo in the first
few days of development, unlike in mice where hemizygous VegfA
mutants show defective vasculogenesis and die between 11 and 12 days
after conception (dpc).3-5 These differences are likely secondary to the
ability of oxygen to diffuse directly into the embryo. Homozygous
cloche embryos, which lack all endothelium and circulating erythro-
cytes do not die until 4 to 5 dpf.31,51,52 These results may also be due to
activity of other molecules, such as Vegfc, that retain activity in these
studies.53 However, these data are consistent with a model in which
VegfAa and VegfAb have functionally diverged since arising from a
common ancestor.
Beginning at 3 dpf, a delay in further maturation was seen in
VegfAb morphants compared with controls (Figure 4C,D). How-
ever, 2 lines of evidence support our contention that the defects in
vascular development were not caused by a general developmental
delay in VegfAb morphants. The first is that the angiogenic defects
were seen in both active VegfAb morpholinos, and not in their 5-bp
mismatch injected controls. The second is that the defects in
angiogenesis were visible at 2 dpf, when no overall differences in
appearance were seen between the morphants and their controls.
We further believe these defects are specific to the loss of VegfAb
functioning in the injected embryos. Injection of equivalent
amounts of 5-bp mismatch morpholinos did not affect vascular
development, and coinjection of the active VegfAb-75 morpholino
that targets the 5ЈUTR upstream of the start codon, with 50 pg each
vegfAb171 and vegfAb210 RNA, reduced the number of embryos with
severe defects in angiogenesis by more than 50% (8/32 vs 22/40
abnormal embryo).
Multiple RTK receptors are required for normal zebrafish
vascular development
Morpholinos targeting both Kdra and Kdrb resulted in defects in
vasculogenesis, especially of the intersegmental arteries, support-
ing a common pathway resulting from RTK activation leading to
embryonic zebrafish vascular development. Yet compared with the
knockout mouse, the phenotype of the combined morphant is less
severe, possibly because of residual activity of the zebrafish flt1
gene, or possibly because other zebrafish genes may have acquired
roles in vasculogenesis and angiogenesis. Lending support to these
possibilities is that zebrafish treated with the type III RTK VEGF
receptor inhibitor PTK787/ZK222584, which blocks all VEGF
receptor activity, lack all major blood vessels.54
An alternative explanation for the differences seen between the
murine KDR knockout and the zebrafish KDR morphant pheno-
types is because of evolutionary changes to the molecular function
of zebrafish RTKs and other genes. For example, the zebrafish
phospholipase C ␥-1 (plc␥1) mutant displays only a subset of the
defects seen in Plc␥1 knockout mice. Significantly, the zebrafish
plc␥1 mutant fails to replicate the complete defect of
erythropoiesis.55,56
The overlapping expression patterns of Kdra and Kdrb, com-
bined with the differential knockdown phenotypes seen in these
studies, suggest that it is not the expression of these RTKs that
generates differential development of the vascular system. We
instead speculate that differences in ligand expression and/or other
not yet described RTKs in the zebrafish account for these differ-
ences. These data are essentially similar to those seen by Covassin
et al in their studies of these 2 RTK receptors.57 In their report, they
described the morphant phenotypes of Kdra and Kdrb and extended
their observations of the interaction between these 2 RTKs to their
differing interactions in the formation of veins and arteries. With
these experiments, we have extended their findings to demonstrate
that multiple Vegfs can bind and activate these 2 RTKs. The
successful in vitro expression and activation of both zebrafish Kdrs
by both VegfA genes support a model in which both VegfA loci
may signal through multiple RTKs. We can further hypothesize that
these interactions may account for some of the complexities of
VegfA signaling seen in developmental and pathological studies.
Transfection of native zebrafish vegfA constructs into mamma-
lian cell lines showed that VegfAa121 and VegfAa165 are secreted,
but VegfAb171 and VegfAb210 are not, even though a well-defined
signal peptide is present. Although it is possible to speculate that
the VegfAb isoforms are secreted by a novel zebrafish-specific
mechanism in vivo, this effect may be due to the nature of zebrafish
signal peptide recognition in a mammalian cell culture system.
Because of the ease with which zebrafish genes can be targeted
and studied, we believe that the zebrafish will make important
contributions to understanding these pathways. In addition, newly
available mutant alleles and chemical inhibitors will enable a more
Figure 6. VegfAb secretion and binding. Full-length vegfAa121, vegfAa165, veg-
fAb171, and vegfAb210 tagged with a V5 epitope at the N-termini were transfected into
COS7 cells. (A) Western blot with a V5 antibody showed that although all 4 proteins
are detectable in cell lysates (B) only VegfAa121 and VegfAa165 were secreted.
(C) Transfected full-length kdra and kdrb fused to a V5 epitope were detectable in cell
lysates of COS7 cells. (D) Replacing the endogenous signal peptide on all isoforms
with the Ig signal peptide resulted in secretion of both VegfAa and VegfAb. HEK 293
cells were transfected individually with Ig-HA-vegfAa165-Myc, Ig-HA-vegfAb171-
Myc, soluble kdra-V5, and soluble kdrb-V5. Transfected cells were grown for 4 days,
and conditioned media were collected, mixed, and incubated overnight. Immunopre-
cipitation (IP) with a V5 antibody (RTKs), blotting with Myc antibody (VegfAs),
demonstrates that soluble Kdra bound to both VegfAa165 and VegfAb171; however,
Kdrb did not appear to bind VegfAb171 as well as VegfAa165 in this assay.
(E) Ig-HA-vegfA-V5 constructs and Kdra-V5/pcDNA3.1 or Kdrb-V5/pcDNA3.1 were
cotransfected into COS7 cells blotted with antiphosphotyrosine antibody, indicating
activation of both RTKs by both VegfAa and VegfAb or (F) anti-V5 antibody
demonstrating expression of the Kdra or Kdrb proteins.
3634 BAHARY et al BLOOD, 15 NOVEMBER 2007 ⅐ VOLUME 110, NUMBER 10
For personal use only.on October 24, 2014.by guestwww.bloodjournal.orgFrom
9. complete block of VegfA signaling and identify the individual
contributions of the different, redundant signal molecules and their
receptors to vasculogenesis and hematopoiesis. As it becomes
increasingly clear that vasculogenesis and angiogenesis act not
only in embryonic development, but also in the adult to modulate
wound healing, tissue regeneration, and progression of human
diseases, the VegfA signaling pathway will become an important
target for new molecular medicines.58-60 Because of its unique
characteristics, the zebrafish system is an excellent system to model
human development and disease and a detailed understanding of
vasculogenesis and angiogenesis in the zebrafish will help make
new medicines in this area a reality.
Acknowledgments
This work was supported in part by grants from the Crohn’s and
Colitis Foundation of America, March of Dimes, National Insti-
tutes of Health (NIH) K08 HL03777 (N.B.), and NIH/National
Cancer Institute 45548 (M.K.).
Authorship
Contribution: N.B. designed and performed research, analyzed
data, and wrote the paper; K.G. designed and performed research
and analyzed data; C.S. performed research, analyzed data, and
wrote the paper; G.W. performed research and analyzed data; J.L.,
C.A.S., and S.S.B. performed research; and M.K. and L.I.Z.
designed research.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Nathan Bahary, University of Pittsburgh,
3501 5th Ave, 5058 BST3, Pittsburgh, PA 15260; e-mail:
bahary@pitt.edu.
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