Jak2 protein knockdown was achieved in BaF3 cells using siRNA nucleofection to study the effects on proliferation dependent on GCSF receptor isoforms. Results showed that Jak2 knockdown halted proliferation of cells expressing isoform IV but not isoform I, validating previous findings of Jak2's essential role in isoform IV signaling. Future experiments are planned to further examine downstream signaling effects of Jak2 knockdown and address issues with cell viability in the absence of Jak2.
Jak2 protein knockdown was achieved in BaF3 cells using siRNA nucleofection to study the effects on proliferation dependent on GCSF receptor isoforms. Results showed that Jak2 knockdown halted proliferation of cells with isoform IV but not isoform I, validating previous findings of Jak2's essential role in isoform IV signaling. Future experiments are planned to further examine downstream effects of Jak2 knockdown and address issues with cell viability in the absence of Jak2.
1. The study examined the effect of nerve growth factor (NGF) and dexamethasone on the proliferation, differentiation, and integrin expression of PC12 cells cultured on collagen and fibronectin coatings.
2. The results showed that PC12 cells proliferated best on collagen before day 7 and on fibronectin past day 7. NGF induced neurite growth on collagen but no differentiation was observed on fibronectin.
3. Expression of alpha-5 and beta-1 integrin subunits decreased from day 1 to day 7 in both 2D and 3D culture conditions.
The document discusses exploring disease networks and how science is performed through networked approaches. It describes how understanding disease requires integrating DNA, RNA, protein and molecular networks. It highlights the EGFR pathway example to show biomarkers can predict treatment response complexity. CETP inhibition example shows causal relationships are not always correlative. Networked approaches are needed to generate, analyze and support new models through data sharing to help understand disease mechanisms and save costs. Synapse is proposed as a platform to enable open sharing of clinical and genomic data as well as model building, similar to how software development occurs through tools like GitHub.
Prevention of lysosomal storage diseases and derivation of2Palaelo
This study describes the use of preimplantation genetic diagnosis (PGD) to prevent the transmission of four lysosomal storage disorders: Tay-Sachs disease, Gaucher disease, Fabry disease, and Hunter syndrome. PGD was performed on 28 embryos from 9 couples, identifying 2 embryos with mutations and deriving stem cell lines from them. The technique was highly accurate, avoiding transmission of diseases while establishing mutant stem cell lines for research.
Prevention of lysosomal storage diseases and derivation of2Palaelo
This study describes the use of preimplantation genetic diagnosis (PGD) to prevent the transmission of four lysosomal storage disorders: Tay-Sachs disease, Gaucher disease, Fabry disease, and Hunter syndrome. PGD was performed on 28 embryos from 9 couples, identifying 2 embryos with mutations and deriving stem cell lines from them. The technique was highly accurate, avoiding transmission of diseases while establishing mutant stem cell lines for research.
1) A pool of peptides extracted from wheat bud chromatin was found to inhibit tumor cell growth by causing defective DNA synthesis.
2) Experiments showed the peptides induced higher levels of DNA fragmentation and replication during early and mid S-phase, but lower replication in late S-phase, indicating defective DNA synthesis.
3) The peptides also decreased DNA synthesis at mid S-phase, activated the DNA damage response pathway, and increased levels of p21 and phospho-p53 proteins, demonstrating a DNA damage response. However, surviving cells were able to progress normally through the cell cycle when removed from the peptides.
This document describes a study using Denaturing Gradient Gel Electrophoresis (DGGE) to differentiate variants of the Infectious Salmon Anemia Virus (ISAv). DGGE allows detection of single point mutations by separating PCR-generated DNA fragments of the same length but different sequence. The study examines variants in segments 5 and 6 of the ISAv genome, which are important for pathogenicity. DGGE analysis of the variants showed distinct banding patterns, demonstrating the technique's ability to distinguish single nucleotide polymorphisms and its potential as a novel alternative for ISAv variant differentiation.
This document analyzes genetic variation at the Pfs48/45 gene and microsatellite loci in 255 Plasmodium falciparum isolates from 5 populations. It finds:
1) Alleles and haplotypes of 5 SNPs in the Pfs48/45 gene varied extremely between populations, much more so than alleles at 11 neutral microsatellite loci.
2) Measurements of between-population allele frequency variation (FST) were 4-7 times higher for Pfs48/45 than microsatellites, both within and between continents.
3) The highly skewed Pfs48/45 patterns suggest divergent selection on the protein's amino acid sequence between populations, indicating it may determine game
Jak2 protein knockdown was achieved in BaF3 cells using siRNA nucleofection to study the effects on proliferation dependent on GCSF receptor isoforms. Results showed that Jak2 knockdown halted proliferation of cells with isoform IV but not isoform I, validating previous findings of Jak2's essential role in isoform IV signaling. Future experiments are planned to further examine downstream effects of Jak2 knockdown and address issues with cell viability in the absence of Jak2.
1. The study examined the effect of nerve growth factor (NGF) and dexamethasone on the proliferation, differentiation, and integrin expression of PC12 cells cultured on collagen and fibronectin coatings.
2. The results showed that PC12 cells proliferated best on collagen before day 7 and on fibronectin past day 7. NGF induced neurite growth on collagen but no differentiation was observed on fibronectin.
3. Expression of alpha-5 and beta-1 integrin subunits decreased from day 1 to day 7 in both 2D and 3D culture conditions.
The document discusses exploring disease networks and how science is performed through networked approaches. It describes how understanding disease requires integrating DNA, RNA, protein and molecular networks. It highlights the EGFR pathway example to show biomarkers can predict treatment response complexity. CETP inhibition example shows causal relationships are not always correlative. Networked approaches are needed to generate, analyze and support new models through data sharing to help understand disease mechanisms and save costs. Synapse is proposed as a platform to enable open sharing of clinical and genomic data as well as model building, similar to how software development occurs through tools like GitHub.
Prevention of lysosomal storage diseases and derivation of2Palaelo
This study describes the use of preimplantation genetic diagnosis (PGD) to prevent the transmission of four lysosomal storage disorders: Tay-Sachs disease, Gaucher disease, Fabry disease, and Hunter syndrome. PGD was performed on 28 embryos from 9 couples, identifying 2 embryos with mutations and deriving stem cell lines from them. The technique was highly accurate, avoiding transmission of diseases while establishing mutant stem cell lines for research.
Prevention of lysosomal storage diseases and derivation of2Palaelo
This study describes the use of preimplantation genetic diagnosis (PGD) to prevent the transmission of four lysosomal storage disorders: Tay-Sachs disease, Gaucher disease, Fabry disease, and Hunter syndrome. PGD was performed on 28 embryos from 9 couples, identifying 2 embryos with mutations and deriving stem cell lines from them. The technique was highly accurate, avoiding transmission of diseases while establishing mutant stem cell lines for research.
1) A pool of peptides extracted from wheat bud chromatin was found to inhibit tumor cell growth by causing defective DNA synthesis.
2) Experiments showed the peptides induced higher levels of DNA fragmentation and replication during early and mid S-phase, but lower replication in late S-phase, indicating defective DNA synthesis.
3) The peptides also decreased DNA synthesis at mid S-phase, activated the DNA damage response pathway, and increased levels of p21 and phospho-p53 proteins, demonstrating a DNA damage response. However, surviving cells were able to progress normally through the cell cycle when removed from the peptides.
This document describes a study using Denaturing Gradient Gel Electrophoresis (DGGE) to differentiate variants of the Infectious Salmon Anemia Virus (ISAv). DGGE allows detection of single point mutations by separating PCR-generated DNA fragments of the same length but different sequence. The study examines variants in segments 5 and 6 of the ISAv genome, which are important for pathogenicity. DGGE analysis of the variants showed distinct banding patterns, demonstrating the technique's ability to distinguish single nucleotide polymorphisms and its potential as a novel alternative for ISAv variant differentiation.
This document analyzes genetic variation at the Pfs48/45 gene and microsatellite loci in 255 Plasmodium falciparum isolates from 5 populations. It finds:
1) Alleles and haplotypes of 5 SNPs in the Pfs48/45 gene varied extremely between populations, much more so than alleles at 11 neutral microsatellite loci.
2) Measurements of between-population allele frequency variation (FST) were 4-7 times higher for Pfs48/45 than microsatellites, both within and between continents.
3) The highly skewed Pfs48/45 patterns suggest divergent selection on the protein's amino acid sequence between populations, indicating it may determine game
This document discusses the marriage of translational medicine and big data. It notes that predicting treatment response to known oncogenes like EGFR is complex and requires detailed understanding of genetic backgrounds. Networks can identify genes causal for disease. The approach uses probabilistic causal network models, with over 80 publications validating the scientific approach. Sage Bionetworks is building disease maps and data repositories through collaborations with industry, foundations, government and academia. Fundamentally, biological science hasn't changed due to omics but iterative networked approaches are needed to generate, analyze and support new disease models.
The document summarizes a study on gene expression during regeneration of Drosophila wing imaginal discs. Microarray analysis found over 1,000 genes differentially expressed between cut and intact discs in the first 24 hours after wounding, indicating a strong initial response to injury. Many genes involved in apoptosis, stress response, cytoskeletal activity and JNK signaling showed increased expression. Between 24-72 hours, expression returned toward normal levels as discs resumed activities interrupted by wounding. The study identifies signaling pathways and gene categories important for disc regeneration.
Clinical grade ex vivo expanded human natural killer (NK) cellslifextechnologies
This document summarizes a study that evaluated a method for expanding clinical-grade natural killer (NK) cells for adoptive transfer immunotherapy. The study found that:
1) NK cells expanded over 4,000-fold when co-cultured with irradiated lymphoblastoid cells over 21 days.
2) The expanded NK cells had increased expression of activating receptors like TRAIL, FasL, and NKG2D compared to resting NK cells.
3) The expanded NK cells showed significantly higher cytotoxicity against tumor cells treated with bortezomib compared to resting NK cells.
4) The expanded NK cells secreted higher levels of cytokines like IFN-γ when co-c
Prevention of lysosomal storage diseases and derivation of2Palaelo
This study evaluated the use of preimplantation genetic diagnosis (PGD) to prevent the transmission of four lysosomal storage disorders (TSD, GD, FD, HS) in couples at risk. PGD was performed on 28 embryos from 8 couples, identifying 2 embryos with mutations for Hunter syndrome and GD. These embryos were used to derive 2 human embryonic stem cell lines expressing markers of an undifferentiated state. PGD accurately diagnosed mutations in embryos and allowed derivation of disease-specific stem cell lines, demonstrating the potential of PGD to avoid inherited diseases while generating disease models.
Prevention of Lysosomal Storage Diseases and Derivation of Mutant StemCell Li...Palaelo
This study used preimplantation genetic diagnosis (PGD) to establish stem cell lines from embryos affected by four lysosomal storage diseases (LSDs): Tay-Sachs disease, Gaucher disease, Fabry disease, and Hunter syndrome. PGD was performed on 329 eggs from 56 cycles involving 20 families with known LSD mutations. Genetic analysis of single blastomeres and polar bodies was used to identify mutations. Two human embryonic stem cell lines were derived - one from a female Hunter syndrome embryo and one from a compound heterozygote embryo for Gaucher disease mutations. The stem cell lines showed typical embryonic stem cell characteristics and normal karyotypes. This demonstrates PGD can avoid transmission of LSD
This study aimed to identify the site of ubiquitination on Activation-Induced Cytidine Deaminase (AID) by the ubiquitin ligase RING Finger Protein 126 (RNF126). Using site-directed mutagenesis, the researchers found that mutating all lysine residues (K0 mutant) prevented ubiquitination, suggesting ubiquitination occurs on a lysine. Analysis of single lysine mutants revealed the K22 mutant still showed ubiquitination, indicating K22 is the main site. However, RNF126 can ubiquitinate AID at other residues as well. Together, this suggests RNF126 favors K22 as the primary ubiquitination site on AID, though it can modify AID at additional
Manipulation of gene expression in prokaryotesSabahat Ali
For expression of gene in a particular vector, always used strong regulatable promoter (lac promoter, trp promoter, tac promoter , trc promoter, pL promoter, T7 gene promoter)
use of dual plasmid system & fusion proteins
How we can increase our protein product yield?
A descoberta da capacidade de certos RNAs serem capazes de bloquear o RNA mensageiro (por serem complementares base a base) há alguns anos,abriu as portas do tratamento de inibição da expressão nucleica de proteínas anormais, responsáveis por doenças. Agora, surgem os miRNA que são produzidos em introns ou exons de gens formadores de proteínas. Estão relacionados ao câncer e diabetes.
Prevention of Lysosomal Storage Diseases and Derivation of Mutant StemCell Li...Palaelo
Preimplantation genetic diagnosis was used to avoid the transmission of four lysosomal storage disorders (TSD, GD, FD, HS) in couples at risk. 329 eggs from 56 cycles underwent PGD and analysis, resulting in 20 families avoiding transmission. Two human embryonic stem cell lines were derived from embryos diagnosed with Hunter syndrome and Gaucher disease. The cell lines expressed stem cell markers and had a normal karyotype. PGD is an effective technique for avoiding genetic transmission of lysosomal storage disorders and generating disease-specific stem cell lines.
This document summarizes research examining the effects of elevated cyclic AMP (cAMP) levels on expression of the chemokine receptor CXCR4 in human hematopoietic progenitor cells. The study finds that increased cAMP, through activation of protein kinase C ζ (PKCζ), leads to higher CXCR4 expression on the cell surface. This enhanced CXCR4 expression results in greater migration, adhesion, survival and homing of progenitor cells. The cAMP/PKCζ pathway interacts with TNFα signaling and converges on CXCR4 expression and secretion of matrix metalloproteinases.
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
This document describes a research project that aimed to sequence the aldolase B gene and discover new mutations that cause hereditary fructose intolerance (HFI). The researchers first attempted to amplify five fragments of the aldolase B gene via polymerase chain reaction (PCR) and analyze the results using gel electrophoresis. Fragment 5 was eventually successfully amplified from a plasmid, but fragments 2 and 4 could not be amplified from patient genomic DNA despite varying PCR conditions. Further optimization is still needed to amplify the fragments from genomic DNA and ultimately sequence the patient DNA to identify new mutations.
Cdc6 knockdown inhibits human neuroblastoma cell proliferationgan-navi
Luo Feng Æ Jerry R. Barnhart Æ Robert C. Seeger Æ Lingtao Wu Æ
Nino Keshelava Æ Sheng-He Huang Æ Ambrose Jong
Received: 19 October 2007 / Accepted: 10 January 2008
Springer Science+Business Media, LLC. 2008
This document summarizes research on the major types of cell death: apoptosis, autophagy, and necrosis. It provides a brief history of the discovery and understanding of each type of cell death. It also describes the key morphological and molecular characteristics of each type of cell death, as well as physiological and pathophysiological functions. The document then discusses experimental approaches to study cell death mechanisms using tools from QIAGEN, including gene expression analysis, epigenetic analysis, and functional studies using reporter assays, siRNA, and shRNA.
Lectut btn-202-ppt-l36. genetic transformation of plantsRishabh Jain
Genetic transformation of plants can occur through several methods, including uptake of foreign DNA by seeds and seedlings, uptake of DNA by pollen, and transformation of protoplasts. Agrobacterium-mediated transformation uses the soil bacterium Agrobacterium tumefaciens or Agrobacterium rhizogenes to transfer DNA into plant cells, while the gene gun method directly transfers DNA particles into cells and tissues. Protoplast transformation involves fusing isolated plant cells without cell walls to foreign organelles, viruses, microorganisms, or isolated DNA to produce genetically transformed plants.
Selection and screening of recombinant clones neeru02
This document discusses several methods for selecting recombinant clones after introducing recombinant DNA into host cells:
- Direct selection involves using a gene from the inserted DNA that confers antibiotic resistance to select clones that grow on media containing that antibiotic.
- Insertional inactivation selection works by inactivating a host gene when foreign DNA inserts into it, allowing selection of recombinants.
- Blue-white screening uses a vector with a disrupted lacZ gene; foreign DNA insertion repairs the gene, allowing recombinants to be identified by colony color.
- Colony hybridization detects recombinants by transferring colonies to a membrane and probing for the inserted DNA sequence.
- Immunological tests identify clones expressing antigens encoded by the
The document discusses the role of MIC-A antibodies in renal allograft loss. It presents the case of a patient whose renal function gradually deteriorated, associated with a weakly positive MICA antibody screen. Biopsies showed glomerulopathy and chronic allograft nephropathy. Based on the positive MICA screen, the patient was treated for possible MICA-related rejection with steroids, which reduced their creatinine level. The document then reviews the expression of MICA antigens and their role in activating NK and T cells through NKG2D engagement. It discusses evidence that MICA antibody mismatch can lead to graft loss and the challenges in detecting non-HLA antibodies.
Este documento describe los desafíos de sostenibilidad que enfrenta Londres y algunas propuestas para abordarlos. En la década de 1980, Londres priorizó las inversiones a corto plazo y la construcción de oficinas, comercios y viviendas, descuidando el espacio público. Esto llevó a un desarrollo insostenible. También ha habido un crecimiento sin límites del parque automotor, lo que segregó el centro y afectó el comercio. La propuesta es recuperar espac
This document discusses the marriage of translational medicine and big data. It notes that predicting treatment response to known oncogenes like EGFR is complex and requires detailed understanding of genetic backgrounds. Networks can identify genes causal for disease. The approach uses probabilistic causal network models, with over 80 publications validating the scientific approach. Sage Bionetworks is building disease maps and data repositories through collaborations with industry, foundations, government and academia. Fundamentally, biological science hasn't changed due to omics but iterative networked approaches are needed to generate, analyze and support new disease models.
The document summarizes a study on gene expression during regeneration of Drosophila wing imaginal discs. Microarray analysis found over 1,000 genes differentially expressed between cut and intact discs in the first 24 hours after wounding, indicating a strong initial response to injury. Many genes involved in apoptosis, stress response, cytoskeletal activity and JNK signaling showed increased expression. Between 24-72 hours, expression returned toward normal levels as discs resumed activities interrupted by wounding. The study identifies signaling pathways and gene categories important for disc regeneration.
Clinical grade ex vivo expanded human natural killer (NK) cellslifextechnologies
This document summarizes a study that evaluated a method for expanding clinical-grade natural killer (NK) cells for adoptive transfer immunotherapy. The study found that:
1) NK cells expanded over 4,000-fold when co-cultured with irradiated lymphoblastoid cells over 21 days.
2) The expanded NK cells had increased expression of activating receptors like TRAIL, FasL, and NKG2D compared to resting NK cells.
3) The expanded NK cells showed significantly higher cytotoxicity against tumor cells treated with bortezomib compared to resting NK cells.
4) The expanded NK cells secreted higher levels of cytokines like IFN-γ when co-c
Prevention of lysosomal storage diseases and derivation of2Palaelo
This study evaluated the use of preimplantation genetic diagnosis (PGD) to prevent the transmission of four lysosomal storage disorders (TSD, GD, FD, HS) in couples at risk. PGD was performed on 28 embryos from 8 couples, identifying 2 embryos with mutations for Hunter syndrome and GD. These embryos were used to derive 2 human embryonic stem cell lines expressing markers of an undifferentiated state. PGD accurately diagnosed mutations in embryos and allowed derivation of disease-specific stem cell lines, demonstrating the potential of PGD to avoid inherited diseases while generating disease models.
Prevention of Lysosomal Storage Diseases and Derivation of Mutant StemCell Li...Palaelo
This study used preimplantation genetic diagnosis (PGD) to establish stem cell lines from embryos affected by four lysosomal storage diseases (LSDs): Tay-Sachs disease, Gaucher disease, Fabry disease, and Hunter syndrome. PGD was performed on 329 eggs from 56 cycles involving 20 families with known LSD mutations. Genetic analysis of single blastomeres and polar bodies was used to identify mutations. Two human embryonic stem cell lines were derived - one from a female Hunter syndrome embryo and one from a compound heterozygote embryo for Gaucher disease mutations. The stem cell lines showed typical embryonic stem cell characteristics and normal karyotypes. This demonstrates PGD can avoid transmission of LSD
This study aimed to identify the site of ubiquitination on Activation-Induced Cytidine Deaminase (AID) by the ubiquitin ligase RING Finger Protein 126 (RNF126). Using site-directed mutagenesis, the researchers found that mutating all lysine residues (K0 mutant) prevented ubiquitination, suggesting ubiquitination occurs on a lysine. Analysis of single lysine mutants revealed the K22 mutant still showed ubiquitination, indicating K22 is the main site. However, RNF126 can ubiquitinate AID at other residues as well. Together, this suggests RNF126 favors K22 as the primary ubiquitination site on AID, though it can modify AID at additional
Manipulation of gene expression in prokaryotesSabahat Ali
For expression of gene in a particular vector, always used strong regulatable promoter (lac promoter, trp promoter, tac promoter , trc promoter, pL promoter, T7 gene promoter)
use of dual plasmid system & fusion proteins
How we can increase our protein product yield?
A descoberta da capacidade de certos RNAs serem capazes de bloquear o RNA mensageiro (por serem complementares base a base) há alguns anos,abriu as portas do tratamento de inibição da expressão nucleica de proteínas anormais, responsáveis por doenças. Agora, surgem os miRNA que são produzidos em introns ou exons de gens formadores de proteínas. Estão relacionados ao câncer e diabetes.
Prevention of Lysosomal Storage Diseases and Derivation of Mutant StemCell Li...Palaelo
Preimplantation genetic diagnosis was used to avoid the transmission of four lysosomal storage disorders (TSD, GD, FD, HS) in couples at risk. 329 eggs from 56 cycles underwent PGD and analysis, resulting in 20 families avoiding transmission. Two human embryonic stem cell lines were derived from embryos diagnosed with Hunter syndrome and Gaucher disease. The cell lines expressed stem cell markers and had a normal karyotype. PGD is an effective technique for avoiding genetic transmission of lysosomal storage disorders and generating disease-specific stem cell lines.
This document summarizes research examining the effects of elevated cyclic AMP (cAMP) levels on expression of the chemokine receptor CXCR4 in human hematopoietic progenitor cells. The study finds that increased cAMP, through activation of protein kinase C ζ (PKCζ), leads to higher CXCR4 expression on the cell surface. This enhanced CXCR4 expression results in greater migration, adhesion, survival and homing of progenitor cells. The cAMP/PKCζ pathway interacts with TNFα signaling and converges on CXCR4 expression and secretion of matrix metalloproteinases.
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
This document describes a research project that aimed to sequence the aldolase B gene and discover new mutations that cause hereditary fructose intolerance (HFI). The researchers first attempted to amplify five fragments of the aldolase B gene via polymerase chain reaction (PCR) and analyze the results using gel electrophoresis. Fragment 5 was eventually successfully amplified from a plasmid, but fragments 2 and 4 could not be amplified from patient genomic DNA despite varying PCR conditions. Further optimization is still needed to amplify the fragments from genomic DNA and ultimately sequence the patient DNA to identify new mutations.
Cdc6 knockdown inhibits human neuroblastoma cell proliferationgan-navi
Luo Feng Æ Jerry R. Barnhart Æ Robert C. Seeger Æ Lingtao Wu Æ
Nino Keshelava Æ Sheng-He Huang Æ Ambrose Jong
Received: 19 October 2007 / Accepted: 10 January 2008
Springer Science+Business Media, LLC. 2008
This document summarizes research on the major types of cell death: apoptosis, autophagy, and necrosis. It provides a brief history of the discovery and understanding of each type of cell death. It also describes the key morphological and molecular characteristics of each type of cell death, as well as physiological and pathophysiological functions. The document then discusses experimental approaches to study cell death mechanisms using tools from QIAGEN, including gene expression analysis, epigenetic analysis, and functional studies using reporter assays, siRNA, and shRNA.
Lectut btn-202-ppt-l36. genetic transformation of plantsRishabh Jain
Genetic transformation of plants can occur through several methods, including uptake of foreign DNA by seeds and seedlings, uptake of DNA by pollen, and transformation of protoplasts. Agrobacterium-mediated transformation uses the soil bacterium Agrobacterium tumefaciens or Agrobacterium rhizogenes to transfer DNA into plant cells, while the gene gun method directly transfers DNA particles into cells and tissues. Protoplast transformation involves fusing isolated plant cells without cell walls to foreign organelles, viruses, microorganisms, or isolated DNA to produce genetically transformed plants.
Selection and screening of recombinant clones neeru02
This document discusses several methods for selecting recombinant clones after introducing recombinant DNA into host cells:
- Direct selection involves using a gene from the inserted DNA that confers antibiotic resistance to select clones that grow on media containing that antibiotic.
- Insertional inactivation selection works by inactivating a host gene when foreign DNA inserts into it, allowing selection of recombinants.
- Blue-white screening uses a vector with a disrupted lacZ gene; foreign DNA insertion repairs the gene, allowing recombinants to be identified by colony color.
- Colony hybridization detects recombinants by transferring colonies to a membrane and probing for the inserted DNA sequence.
- Immunological tests identify clones expressing antigens encoded by the
The document discusses the role of MIC-A antibodies in renal allograft loss. It presents the case of a patient whose renal function gradually deteriorated, associated with a weakly positive MICA antibody screen. Biopsies showed glomerulopathy and chronic allograft nephropathy. Based on the positive MICA screen, the patient was treated for possible MICA-related rejection with steroids, which reduced their creatinine level. The document then reviews the expression of MICA antigens and their role in activating NK and T cells through NKG2D engagement. It discusses evidence that MICA antibody mismatch can lead to graft loss and the challenges in detecting non-HLA antibodies.
Este documento describe los desafíos de sostenibilidad que enfrenta Londres y algunas propuestas para abordarlos. En la década de 1980, Londres priorizó las inversiones a corto plazo y la construcción de oficinas, comercios y viviendas, descuidando el espacio público. Esto llevó a un desarrollo insostenible. También ha habido un crecimiento sin límites del parque automotor, lo que segregó el centro y afectó el comercio. La propuesta es recuperar espac
L’European Junior Doctors Permanent Working Group (EJD-PWG) est l’organisation médicale en charge de représenter les Jeunes Médecins Européens. Au cours du dernier quart de siècle, l’EJD est intervenu activement dans la défense des professions médicales en Europe avec pour objectif la contribution au développement des travaux, l’éducation et la formation des médecins Juniors.
L’Union Européenne des Médecins Spécialistes (UEMS) est l’organisation médicale représentant les médecins spécialistes en Europe. Elle est composée de Conseils, Sections, Boards et du Conseil Européen d’Accréditation de la Formation Médicale Continue (EACCME). Ses principales compétences relèvent du domaine de la formation post-doctorale (3ème cycle), du développement professionnel continu et de la qualité des soins spécialisés.
La collaboration entre l’EJD et l’UEMS existe depuis plusieurs années sous la forme d’accords formels et informels au prot de la qualité de la formation médicale des médecins Juniors Européens.
reseauprosante.fr
Carol Natalia Osorio Quintero Estudiante de tercer semestre Universidad Ponti...KarolOsorio18
This document summarizes a study that evaluated the anticancer and apoptotic activity of phospholipid extract from Scomberomorus niphonius bone in the SK-N-SH neuroblastoma cell line. The study involved cell viability assays, protein extraction and western blotting, reverse transcriptase PCR, DNA fragmentation, and analysis of results. Neuroblastoma is a cancer that originates from immature nerve cells and is the most common cancer in children under one year old. The conclusions were that molecular biology enables searching for therapeutic alternatives for neuroblastoma and knowledge of molecular processes allows detecting and working on cellular alterations precisely.
1) The document describes a process for deriving neural stem cells from human embryonic stem cells and induced pluripotent stem cells. The stem cells are differentiated into neural progenitor cells and neural stem cell monolayers.
2) An in vitro study was conducted exposing the derived neural stem cells to conditioned media from polymorphonuclear neutrophils and macrophages to study the effects on cell fate. Preliminary results found the conditioned media affected expression of markers for neurons and astrocytes differently between the two neural stem cell lines.
3) Future work is planned to test the stem cells in vivo after spinal cord injury, including assessing cell engraftment, migration, and differentiation patterns. Functional recovery will also be evaluated using various
The document discusses strategies for xenotransplantation using genetically modified pigs. It notes that over 18 patients die each day waiting for organ transplants. Pigs are a promising donor species due to anatomical similarities to humans. However, immunological barriers like hyperacute and acute cellular rejection must be overcome. Methods discussed include genetically knocking out GGTA1 and CMAH genes to eliminate antigen epitopes and introducing human complement regulatory genes. Genome editing tools like ZFNs, CRISPR-Cas9, and SCNT are used to generate multitransgenic pigs lacking immunogenic antigens and capable of preventing rejection. Studies produced GGTA1 knockout piglets and fetuses lacking the alpha-Gal epitope using these techniques.
This document summarizes research on NF-kB signaling in cancer, specifically gastric cancer. It describes the background, structure and activation processes of NF-kB. Experiments show that NF-kB1 and RELA are upregulated in gastric cancer cell lines and tumors, and their knockdown inhibits tumor growth in vitro and in vivo. Furthermore, miR-508-3p is identified as a tumor suppressor that directly targets and downregulates NF-kB1 in gastric cancer. Re-expression of NF-kB1 partly reverses the tumor suppressive effects of miR-508-3p overexpression. In conclusion, downregulation of miR-508-3p contributes to canonical NF-kB activation in gastric tumorigenesis.
1) A Yersinia effector protein called YopJ is known to induce apoptosis in infected macrophages by inhibiting host immune signaling pathways. YopJ from Y. pestis KIM (YopJKIM) has enhanced ability to cause apoptosis and activate caspase-1 compared to other YopJ isoforms.
2) This study found that two amino acid substitutions (F177L and K206E) in YopJKIM are responsible for its increased activity. YopJKIM more strongly inhibits the NF-kB and MAPK pathways than other isoforms.
3) Infection of IKKb-deficient macrophages with Y. pestis activated caspase-1, indicating NF-kB normally suppresses
Tumor suppression and inflammation: controlling the senescence associated se...adamfreund
This is the powerpoint presentation from a talk I gave at a conference in October, 2009. It will be hard to follow without the spoken part, but it will hopefully give anyone who is interested a brief introduction to my thesis research.
This study examined how glioblastoma tumors influence microglia cells' expression of IL-1B and IL-1RA genes through secreted factors. Results showed that glioma cell conditioned media induced both IL-1B and IL-1RA expression in microglia. Inhibiting CSF-1R or CCR1 signaling in microglia enhanced IL-1B expression while repressing IL-1RA, suggesting glioma signals cause an anti-inflammatory microglia phenotype. Future work will assess how these changes in IL-1 family expression impact microglia polarization.
An allelic variant of mTOR leads to decreased DNA damage response in mouse embryonic fibroblasts. The variant allele, R628C, is found in BALB/c mice and results in a single amino acid substitution in the mTOR protein. Mice with the variant allele (KI mice) have decreased survival after radiation exposure and their embryonic fibroblasts show greater DNA damage, increased proliferation, and lower levels of the cell cycle inhibitor p27 compared to wild type mice. The results suggest the mTOR variant renders cells more susceptible to DNA damage and less able to repair it or arrest the cell cycle after radiation.
Cloning and subcloning are techniques used in molecular biology. Cloning involves isolating a gene and inserting it into a vector for propagation. Subcloning moves a gene from one vector to another. The document discusses the history of cloning, types of cloning including DNA cloning, reproductive cloning and therapeutic cloning. It provides details on the basic steps for cloning such as isolation of the gene, insertion into a vector, transformation, identification and expression of the cloned gene. Subcloning involves using restriction enzymes to excise a gene from one vector and ligate it into another vector. The document outlines the procedure and applications of both cloning and subcloning techniques.
This document summarizes an experiment to derive tissue culture cell lines that secrete antibodies against sheep red blood cells (SRBCs). Mouse myeloma cells were fused with spleen cells from mice immunized with SRBCs. The resulting hybrids were selected in HAT medium, and clones that secreted anti-SRBC antibodies were identified using plaque assays. Figures 1 and 2 show that the hybrids expressed antibodies from both parental lines and secreted antibodies that could lyse SRBCs. Figure 3 demonstrates antibody secretion patterns on isolectric focusing, while Figure 4 shows inhibition of lysis by anti-IgM antibody. This technique demonstrated the ability to produce predefined antibodies using fused myeloma-spleen cell lines.
This document discusses the hypothesis that the small ubiquitin-like modifier (SUMO) modification of IκBα, an inhibitor of the NF-κB transcription factor, results in its localization in the nucleus where it functions as a synergy control factor. The hypothesis is that SUMO-IκBα localizes to the nucleus and creates a dynamic nuclear pool that inhibits NF-κB-dependent transcription. A series of experiments are proposed to test this hypothesis, including examining the localization and dynamics of SUMO-IκBα in primary non-transformed cells using cellular fractionation, pulse-chase assays, and FRET.
Immune response in FHM cells following infection with frog virus 3mgray11
This document summarizes a study that examined the differential expression of immune-related genes in fathead minnow cells following infection with frog virus 3 (FV3) or an attenuated 18K knockout mutant. Microarray analysis found that numerous innate and adaptive immune genes were upregulated after wild-type FV3 infection. However, the 18K mutant led to lower expression levels of these genes. This suggests that quantitative differences in gene expression may explain the 18K mutant's attenuated phenotype in vivo. Future studies aim to further analyze host responses to wild-type and mutant viruses to identify key cellular factors involved in protective immunity.
The document outlines six hallmarks of cancer cells:
1. Self-sufficiency in growth signals through autocrine or paracrine signaling and expression of growth-promoting extracellular matrix receptors.
2. Insensitivity to antigrowth signals through mutations that block cell cycle inhibitors like Rb or induce differentiation.
3. Evading apoptosis by mutations in genes like p53 that derail cell suicide pathways and activate pro-survival signals.
4. Limitless replicative potential enabled by mechanisms like telomerase that maintain telomere length indefinitely.
5. Sustained angiogenesis driven by factors like VEGF that promote growth of blood vessels.
6. Tissue invasion and
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.
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.
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.
Activation of NRF-2 blocks HIV and apoptosis in macrophagesJuanPisVelasquez
The document discusses NRF-2, a negative regulator of oxidative stress and inflammation. It travels to the nucleus and binds antioxidant elements, decreasing inflammatory cytokine levels. The study aimed to determine if inhibiting oxidative stress could control HIV replication and improve cell survival. Methods used included PCR, ELISA, Western blotting, and flow cytometry. The conclusions were that these molecular biology techniques helped understand NRF-2's importance, and that NRF-2 activation promotes macrophage survival after HIV infection, aiding patient diagnosis.
This study investigates how inflammatory breast cancer (IBC) cells evade anoikis, a form of programmed cell death triggered by detachment from the extracellular matrix (ECM). The researchers found that two IBC cell lines, KPL-4 and SUM149, resisted anoikis and survived when detached from ECM. Knocking down ErbB2 in KPL-4 cells or EGFR in SUM149 cells increased anoikis and decreased anchorage-independent growth. The ERK/MAPK pathway operated downstream of ErbB2/EGFR to protect the IBC cells from anoikis. Unlike in other cell types, inhibiting ERK did not increase levels of the pro-ap
Survivin is a recently discovered protein that is implicated in controlling cell proliferation and apoptosis. It is overexpressed in most human cancers. Survivin belongs to the inhibitor of apoptosis family and has a unique structure with a single baculovirus IAP repeat. It functions to inhibit caspase activity and apoptosis. Survivin is regulated through the cell cycle and peaks in expression during mitosis, localizing to microtubules of the mitotic spindle. Due to its role in inhibiting apoptosis and promoting cell proliferation, survivin may provide insights into cancer diagnosis and treatment.
Parallel mechanisms of epigenetic reprogramming in the germlineLoki Stormbringer
Germ cells possess the extraordinary and unique capacity to give rise to a new organism and create an enduring link between all generations. To acquire this property, primordial germ cells (PGCs) transit through an unprecedented programme of sequential epigenetic events that culminates in an epigenomic basal state that is the foundation of totipotency. This process is underpinned by genome-wide DNA demethylation, which may occur through several overlapping pathways, including conversion to 5-hydroxymethylcytosine. We propose that the epigenetic programme in PGCs operates through multiple parallel mechanisms to ensure robustness at the level of individual cells while also being flexible through functional redundancy to guarantee high fidelity of the process. Gaining a better understanding of the molecular mechanisms that direct epigenetic reprogramming in PGCs will enhance our ability to manipulate epigenetic memory, cell-fate decisions and applications in regenerative medicine.
Parallel mechanisms of epigenetic reprogramming in the germline
Poster presentation proposal
1. Jak2 Protein Knockdown in BaF3 Cells Causes Differences in Proliferation Dependent on
the Growth Colony Stimulating Factor Receptor Isoforms
M Quinn, H Mehta, SJ Corey
Robert H. Lurie Comprehensive Cancer Center
Northwestern Feinberg School of Medicine
Loyola University Chicago
Abstract Methodology Discussion
Granulocyte colony stimulating factor (G-CSF) is a hormone that Our main method for studying Jak2 protein knockdown was Our findings with the original HGPRT control determined that
has been recognized to play a role in the differentiation and through the use of siRNA nucleofection. 300 nM concentration of siRNA was optimal – though it still did
proliferation of hematopoietic stem cells into neutrophils via the not reach expected knockdown. We expected greater than 90%
G-CSF Receptor. Of the known isoforms of this receptor, Class I is knockdown with this control, and we were only able to achieve
the most common in healthy individuals. However, the Class IV roughly 75% knockdown. However, it is sufficient for our
isoform, which is shorter in the internal cellular domain, shows experimental purposes, and we will continue to use 300 nM as
increased expression in individuals with severe congenital our standard nucleofection concentration.
neutropenia (SCN), acute myeloid leukemia (AML), as well as
individuals who respond poorly to chemotherapy. The Our Jak2 post-nucleofection western blot results lead us to
mechanism by which the Class IV isoform signaling differentiates visually conclude that siRNA duplexes #2 & #4 yield the best
from Class I is not yet known. Previous research has indicated a knockdown in BaF3 HA-tagged cells. For future
Jak2-dependence within the internal signaling of Class IV that is experiments, these are the two duplexes which we will use in
not present in Class I. Through the use of a Jak2 siRNA both GR-I and GR-IV cell types.
nucleofection knockdown, we determined Class IV proliferation
is dependent on the GCSF-Receptor binding to Jak2; without
which, the cells cease to thrive. Our results validate previous Creates temporary block of protein translation through mRNA
findings that Jak2 is essential for Class IV proliferation but has no cleavage. Future Directions
significant effect on Class I. We intend to further examine the
downstream signaling effects of Jak2 through this knockdown o Using both Baf3 HA-GRI and HA-GRIV cells, we nucleofected o Further experiments are necessary to test the effects of Jak2
technique in future studies. 2x106 cells/well using Thermo Scientific Dharmacon siRNA knockdown on cell proliferation in both GCSF-R I and GCSF-R
duplex #2 and #4 (specific to Jak2). IV BaF3 cells. We would like to do this using both cell viability
o After the nucleofection, we incubated the cells in 1.5 ml of as well as MTT assays.
BaF3 media +serum (including mIL3). o Additionally, experiments to validate the downstream effects
Introduction o We collected cell lysates at 48 hours post-nucleofection of Jak2 on Shp2 found in our previous research are desired.
to do western blot analyses.
o A concern of ours is the murine interleukin-3 (mIL-3) serum
The Growth Colony Stimulating Factor Receptor (GCSF-R)
dependency of our BaF3 cells. mIL-3 serum supplement is
influences hematopoietic stem cell differentiation, We used a mouse monoclonal antibody to blot for Jak2 required in BaF3 media, or high cell death will result.
proliferation, and survival. It has previously been determined protein presence, and a rabbit polyclonal Actin antibody for However, mIL-3 requires protein Jak2 in order to be
that there are several different isoforms of this receptor our control. integrated into the cell for effectiveness. Because we are
present in humans. Isoform I is the most prevalent type knocking down Jak2, there is the fear that those cells that are
found in healthy bodies, whereas the truncated Isoform IV successfully nucleofected with siRNA will not survive because
has been found to be present in higher ratios in patients they will be unable to incorporate mIL-3 into the cell. We are
Results
with Severe Congenital Neutropenia (SCN) and Acute currently troubleshooting this issue prior to further
Myeloid Leukemia (AML). In vitro studies have previously A qPCR of HGPRT as control was performed to determine experimentation.
shown that truncated GCSF-R IV causes increased nucleofection efficiency at different siRNA concentrations:
proliferation, with no effective differentiation in myeloid
precursor cells. References
o Mehta HM, Muneyoshi F, Glaubach T, Lee DW, Andolina
JR, Whichard Z, Quinn MP, Kao W, Sarkar CA, Maciejewski
JP, Corey SJ. Leukemia-associated loss of the GCSF receptor c-
terminus alters growth and differentiation properties and Jak-
Stat signaling. Blood (submitted 10/30/2012)
Differentiation o Weigert O, Lane A, Bird L, et al. Genetic resistance to JAK2
enzymatic inhibitors is overcome by HSP90 inhibition. J Exp
Neutrophil Med. 2012; 209(2):259-273. DOI 10.1084/jem.20111694.
Monocytes
Proliferation
Lymphocytes
Survival Acknowledgements
A western blot analysis was done after the original nucleofection
to determine whether the siRNA was effective in knocking down I would like to thank the Corey Lab at Northwestern Feinberg for
Jak2 at 300 nM concentration (and which ones are most giving me this opportunity.
effective):
The differences in internal signaling pathways between
isoforms I and IV are of critical importance, as they could be
potential drug targets in future treatments. siRNA 0 Scr #1 #2 #3 #4
Jak2
Our previous research has shown a differential response to
Actin
the protein Jak2, which has a dose-dependent downstream
effect on protein Shp-2 in GCSF-R IV but not GCSF-R I. For BaF3 HA-GRI 48 Hr Post-Nucleofection
this reason, knockdown studies of Jak2 in both cell-receptor
types could provide an important avenue for researching the
differences between these two receptors.