A method was developed to isolate genomic clones from a cosmid library using homologous recombination in vivo. This method was used to isolate the human interleukin 2 (IL2) gene. A cosmid library was packaged into lambda phage particles in vivo. A host strain carrying IL2 cDNA sequences was infected with the packaged cosmid library. Recombinants containing antibiotic resistance genes from both vectors were selected. A cosmid clone containing the chromosomal IL2 gene was identified. After transferring the gene into mouse cells, human IL2 was expressed constitutively.
Universal and rapid salt extraction of high quality genomic dna for pcr-based...CAS0609
This document describes a simple and universal method for extracting high-quality genomic DNA from a variety of organisms including plants, fungi, insects, and shrimp. The method uses a salt-based homogenizing buffer and SDS to extract DNA from as little as 50mg of fresh tissue. The extracted DNA is of sufficient quality and quantity to be used in PCR, restriction digestion, and other molecular techniques. The method is fast, inexpensive, and does not require expensive equipment, making it suitable for laboratories with limited resources. Test results demonstrated the method successfully extracted high molecular weight DNA from many diverse organisms without modification, indicating its universal applicability.
This study demonstrated a novel natural transformation mechanism in Actinobacillus actinomycetemcomitans (A.a.) that is independent of uptake signal sequences and the Tfox gene. The study showed that A.a. could be transformed with genomic and plasmid DNA present in microvesicles secreted into the growth medium of donor cells. This transformation occurred both in the presence and absence of components normally required for natural transformation in A.a. The results suggest outer membrane adhesion and fusion of donor microvesicles with recipient cells allows DNA delivery and homologous recombination. This novel mechanism could provide an easier method for genetically transforming A.a. compared to conventional techniques.
The document discusses genome evolution in plants, with a focus on wheat and its wild relatives. It describes how:
- Wheat has a very large and repetitive genome compared to other plants like Arabidopsis.
- Repetitive DNA sequences play an important role in genome and chromosome evolution, and can be used to track chromosomes.
- Wild relatives like Aegilops ventricosa have been used in chromosome engineering to introduce useful traits like disease resistance to wheat.
- Molecular cytogenetics techniques allow studying the inheritance and distribution of repetitive sequences during speciation and hybridization.
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 summarizes work done on the Japanese Encephalitis Virus (JEV) from December 2009 to November 2014. Key findings include:
1) Expression and purification of the JEV envelope domain 3 (ED3) protein, which mediates viral attachment to host cells. Antibodies were generated against ED3.
2) Studies using cell surface biotinylation to identify potential ED3 receptor molecules on porcine kidney and neuroblastoma cells. Interactions were observed between ED3 and proteins from infected neuroblastoma cells.
3) Expression of JEV non-structural protein NS5 and purification under native conditions, yielding 15mg/L of protein.
4) Attemp
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1YArunkumar K.R.
Structure of a carotenoid gene
cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene
by functional complementation in Escherichia coli.
This document describes a study that generated a synthetic antibody library with predefined complementarity determining regions (CDRs) for high-throughput antibody selection. The library was constructed using oligonucleotides encoding designed CDR sequences synthesized on microarrays. The library was used to select novel antibodies against four human protein targets. Enriched CDR sequences from early selection rounds were identified and reconstructed to generate a consensus antibody specific for each target.
Universal and rapid salt extraction of high quality genomic dna for pcr-based...CAS0609
This document describes a simple and universal method for extracting high-quality genomic DNA from a variety of organisms including plants, fungi, insects, and shrimp. The method uses a salt-based homogenizing buffer and SDS to extract DNA from as little as 50mg of fresh tissue. The extracted DNA is of sufficient quality and quantity to be used in PCR, restriction digestion, and other molecular techniques. The method is fast, inexpensive, and does not require expensive equipment, making it suitable for laboratories with limited resources. Test results demonstrated the method successfully extracted high molecular weight DNA from many diverse organisms without modification, indicating its universal applicability.
This study demonstrated a novel natural transformation mechanism in Actinobacillus actinomycetemcomitans (A.a.) that is independent of uptake signal sequences and the Tfox gene. The study showed that A.a. could be transformed with genomic and plasmid DNA present in microvesicles secreted into the growth medium of donor cells. This transformation occurred both in the presence and absence of components normally required for natural transformation in A.a. The results suggest outer membrane adhesion and fusion of donor microvesicles with recipient cells allows DNA delivery and homologous recombination. This novel mechanism could provide an easier method for genetically transforming A.a. compared to conventional techniques.
The document discusses genome evolution in plants, with a focus on wheat and its wild relatives. It describes how:
- Wheat has a very large and repetitive genome compared to other plants like Arabidopsis.
- Repetitive DNA sequences play an important role in genome and chromosome evolution, and can be used to track chromosomes.
- Wild relatives like Aegilops ventricosa have been used in chromosome engineering to introduce useful traits like disease resistance to wheat.
- Molecular cytogenetics techniques allow studying the inheritance and distribution of repetitive sequences during speciation and hybridization.
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 summarizes work done on the Japanese Encephalitis Virus (JEV) from December 2009 to November 2014. Key findings include:
1) Expression and purification of the JEV envelope domain 3 (ED3) protein, which mediates viral attachment to host cells. Antibodies were generated against ED3.
2) Studies using cell surface biotinylation to identify potential ED3 receptor molecules on porcine kidney and neuroblastoma cells. Interactions were observed between ED3 and proteins from infected neuroblastoma cells.
3) Expression of JEV non-structural protein NS5 and purification under native conditions, yielding 15mg/L of protein.
4) Attemp
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1YArunkumar K.R.
Structure of a carotenoid gene
cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene
by functional complementation in Escherichia coli.
This document describes a study that generated a synthetic antibody library with predefined complementarity determining regions (CDRs) for high-throughput antibody selection. The library was constructed using oligonucleotides encoding designed CDR sequences synthesized on microarrays. The library was used to select novel antibodies against four human protein targets. Enriched CDR sequences from early selection rounds were identified and reconstructed to generate a consensus antibody specific for each target.
This master's dissertation aimed to demonstrate gene expression in Rat1 fibroblast cells transformed by EVI1 and the relationship between EVI1 levels and CAIII gene expression. Real-time PCR and western blotting showed higher CAIII gene and protein expression in Rat1neo cells compared to Rat15.6 cells, which overexpress EVI1. Luciferase assays also demonstrated higher activity in Rat1neo cells, indicating higher CAIII expression. Silencing CAIII in Rat1neo cells increased caspase 3 activity after hydrogen peroxide treatment, showing CAIII protects against apoptosis. The results suggest EVI1 overexpression represses CAIII expression, reducing protection against oxidative stress. Therefore, oxidative stress agents may selectively target cancer cells overexpressing
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).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
1. The study analyzed 30 Vibrio cholerae O1 isolates from patients in Delhi, India from 2012-2014 using mismatch amplification mutation assay (MAMA) PCR to identify the ctxB genotype.
2. All isolates were biochemically identified as V. cholerae El Tor serotype Ogawa. MAMA PCR results showed that all isolates carried the ctxB gene of the classical biotype, though they were phenotypically El Tor.
3. This identifies an "altered El Tor" variant circulating in Delhi that displays characteristics of both classical and El Tor biotypes in possessing the classical ctxB gene despite being phenotypically El Tor.
This document summarizes research on the Scr74 gene family in Phytophthora infestans and how different variants of this gene are recognized in potato genotypes. The key points are:
1. 27 variants of the Scr74 effector gene were identified from 12 P. infestans isolates. 4 novel variants were cloned.
2. 13 amino acid sites in Scr74 were found to be under positive diversifying selection, indicating these sites are important for pathogen recognition.
3. 17 Scr74 variants were screened using PVX assays on 12 potato genotypes. Some genotypes had strong cell death responses to specific Scr74 variants, suggesting recognition of these variants.
4. Scr74 variants Scr74-A
This study investigated the role of the cellular protein annexin 2 (Anx2) in human immunodeficiency virus type 1 (HIV-1) particle production and infectivity. The researchers knocked down Anx2 expression by 98% in various cell types using lentiviruses encoding short hairpin RNAs against Anx2. They found that knocking down Anx2 did not reduce HIV-1 virus-like particle production in COS-1, 293T, Jurkat T cells, or primary human macrophages. Similarly, murine cells lacking Anx2 produced normal levels of particles. However, supernatants from Anx2-depleted primary human macrophages exhibited decreased infectivity, indicating Anx2 plays a
The document provides information about midterm exams and extra office hours for a class. It announces that the first midterm exam will be on Thursday, February 15 at 6pm in room 155 Dwinelle, and that review sessions will be held during the regular class time that day. It also notes that the professor will not have office hours on February 13.
Boronated Cetuximab CCR tumor targeting in BNCTkent.riley
This document describes a study evaluating boronated cetuximab (BD-C225) for boron neutron capture therapy (BNCT) of epidermal growth factor receptor (EGFR) positive gliomas. In vitro, BD-C225 showed preferential uptake in EGFR positive glioma cells compared to EGFR negative cells. In vivo, rats with EGFR positive glioma tumors received intracerebral BD-C225, achieving high boron levels in the tumors. BNCT with BD-C225 alone or combined with boronophenylalanine extended survival compared to controls. The results provide support for using molecularly targeted boron delivery agents like BD-C225 for BNCT of brain tumors.
- The document discusses research on the cotton root-knot nematode (Meloidogyne incognita), an endoparasitic nematode that infects thousands of plant species and causes billions in crop damage worldwide.
- The research uses Arabidopsis thaliana plants, including mutant strains, to study the nematode's infection process. Mutant plants have mutations in an ABC transporter gene (At4g15320) involved in secreting chemicals that nematodes use to locate host plants.
- The goal is to see if the mutant transporter gene affects nematode infection rates compared to wild-type Arabidopsis. Results found the mutant was actually not a true mutant and showed similar nematode infection to the
The document summarizes research on expressing HIV-1 antigens in plants as a potential vaccine. The researchers found that transient expression in plants is a viable alternative to transgenic plants. They determined that targeting the HIV-1 Gag protein Pr55Gag and its derivative p17/24 to chloroplasts yielded the highest levels of these proteins. Mice vaccinated with p17/24 produced from chloroplasts developed strong CD4+ and CD8+ T cell responses and HIV-specific antibodies, showing it is a promising vaccine candidate.
This document describes research on cloning and expressing the domain III region of the dengue virus envelope protein (DENV EDIII) from serotypes 3 and 4. Key findings include:
1) DENV EDIII from serotypes 3 and 4 were successfully cloned and expressed in E. coli, and the recombinant proteins were purified using nickel affinity and size exclusion chromatography.
2) The purified recombinant EDIII proteins were able to fold correctly and form stable monomers, as shown through analytical methods like SDS-PAGE and gel filtration chromatography.
3) In mouse studies, immunization with the recombinant EDIII proteins, especially when combined with adjuvants, induced strong humoral and cellular immune responses as measured by
- A single episomal plasmid containing Oct4, Sox2, KLF4, L-Myc driven by a hybrid CAG promoter can reprogram human fibroblasts into induced pluripotent stem cells (iPSCs).
- The inclusion of 5 small molecules that enhance reprogramming results in the appearance of proto-colonies by 7 days and mature colonies by 21 days under low oxygen conditions.
- The iPSCs express pluripotency markers, can differentiate into neurons and cardiomyocytes, and grow robustly in a new xeno-free media. Further work will examine residual vector presence and gene expression profiles.
This study aims to generate fusion-defective mutants of Chlamydomonas reinhardtii through random insertional mutagenesis using glass bead transformation. Transformed cells expressing paromomycin resistance are selected and screened for inability to mate. Several mating-deficient clones have been identified that are defective in swimming, agglutination or both. Further analysis will determine if any clones possess a fusion defect and identify the insertion location to study genes involved in fusion.
This study investigated the role of the transcriptional regulator ToxR in biofilm formation and motility in the pathogenic bacterium Vibrio parahaemolyticus. The study found that:
1) A V. parahaemolyticus mutant lacking ToxR showed decreased biofilm formation and reduced swarming and swimming motility compared to the wild type strain.
2) The ToxR regulator was required for rugose colony formation and expression of genes involved in exopolysaccharide production, which are important for biofilm formation.
3) ToxR did not regulate switching between opaque and translucent colony types in V. parahaemolyticus, which is associated with capsular polysaccharide production.
PRODUCTION OF SEROTYPE 6-DERIVED RECOMBINANT ADENO-ASSOCIATED VIRUS IN SERUM-...Dr. Érica Schulze
This document describes research into producing recombinant adeno-associated virus (rAAV) using serum-free suspension cultures of HEK 293 cells. The researchers investigated the effects of different DNA, polyethyleneimine (PEI) transfection reagent, and cell concentrations on rAAV yields. They found that transfecting at a cell concentration of 3x10^6 cells/mL resulted in a 2.5-3.5 fold increase in infectious virus particles (IVP) and genomic particles (Vg) compared to 1x10^6 cells/mL. When testing different PEI:DNA ratios, IVP ranged from 1.19x10^8 to 1.03x10^9 IVP/
A panel of recombinant monoclonal antibodies against zebrafishShahnaz Yusaf
This document describes the development of 10 recombinant monoclonal antibodies against neural receptors and secreted proteins in zebrafish. The antibodies were generated by expressing the extracellular domains of the target proteins in mammalian cells and using them as antigens. The antibodies were characterized, cloned into expression plasmids, and shown to specifically stain their antigens in fixed zebrafish embryo tissues. The staining patterns matched the known expression patterns of the target genes, demonstrating these antibodies will be useful tools for studying neural development in zebrafish.
Poster - Development of double stand break assessment assay with HCS by using...HCS Pharma
The creation of a double strand break (DSB) is accompanied by the phosphorylation of histone H2AX. The measurement of serine 139 phosphorylated histone H2AX (γH2AX) is reported to be a marker of interest to identify potential genotoxic activity.
In order to evaluate the High Content Screening for γH2AX detection, 4 non genotoxic compounds and 9 genotoxic compounds from the ECVAM list I or II were selected to to be tested on HepG2 cell line. These cells offer the advantage to have H2AX expression data in the literature. In parallel, Human primary keratinocytes were included. Indeed these cells would be relevant for investigating skin adverse effects of topical applied xenobiotics with the advantage of High content imaging as valuable tool for screening in the early discovery phase.
This document describes research into using the recombinant dense granule antigen GRA7 from Toxoplasma gondii for serodiagnosis of toxoplasmosis. The GRA7 gene was amplified from T. gondii genomic DNA and inserted into an expression vector. The recombinant GRA7 protein was expressed in E. coli and purified. Western blot analysis showed human sera reacted with the 29 kDa rGRA7 antigen. ELISA tests using rGRA7 showed sensitivity of 96% for IgM detection and 89% for IgG detection, with specificity of 90% for both, demonstrating its potential usefulness for toxoplasmosis diagnosis.
This Masters thesis defense presentation summarizes research identifying MH class IIβ alleles that may confer resistance or susceptibility to Bacterial Cold Water Disease (BCWD) in rainbow trout. The study genotyped MH class IIβ alleles in six families of trout that had been experimentally infected with the BCWD pathogen. Results found five alleles present among the families. The DAB*1001/DAB*0801 genotype had the lowest hazard ratio, suggesting an association with resistance to BCWD, although results were not statistically significant. Future work could examine MH class I alleles and innate immunity genes to better understand resistance in trout.
2004 paper primer ctx dissemination of ctx m-type -lactamases among clinical ...JairAlexanderTllez
This document summarizes a study analyzing 19 clinical isolates of Enterobacteriaceae (16 E. coli and 3 K. pneumoniae) collected from four hospitals in Paris, France between 2000-2002 that exhibited resistance to extended-spectrum cephalosporins. Testing found the resistance was due to various CTX-M-type beta-lactamase enzymes, predominantly CTX-M-15. Most isolates produced both TEM-1 and CTX-M enzymes. The blaCTX-M genes were located on large plasmids and often upstream of the insertion sequence ISEcp1. Five isolates had identical plasmid fingerprints, suggesting clonal dissemination of CTX-M-15-producing strains in
This document summarizes research investigating the biosynthesis and processing of succinate dehydrogenase (SDH) subunits in cultured mammalian cells. The key points are:
1. Antisera were produced against purified bovine heart SDH and its large and small subunits, which detected precursor and mature forms of the subunits in rat, pig, and bovine cell lines.
2. In pig kidney cells, newly synthesized precursors of the large and small SDH subunits were detected that were 1000-2000 and 4000-5000 Da larger than the mature forms, respectively.
3. Pulse-chase experiments showed the precursor forms were fully processed to the mature subunits within 45 minutes when uncouplers of
This paper describes a regulatory pathway controlling expression of Borrelia burgdorferi OspC and DbpA proteins. The study found that in B. burgdorferi strain 297, the alternative sigma factor RpoN controls expression of the alternative sigma factor RpoS. RpoS then governs expression of the outer surface lipoproteins OspC and DbpA. This regulatory network was determined through targeted gene disruption of rpoN and rpoS, followed by genetic complementation. The findings provide insight into key regulatory networks that impact B. burgdorferi pathogenesis, host range, and virulence.
This master's dissertation aimed to demonstrate gene expression in Rat1 fibroblast cells transformed by EVI1 and the relationship between EVI1 levels and CAIII gene expression. Real-time PCR and western blotting showed higher CAIII gene and protein expression in Rat1neo cells compared to Rat15.6 cells, which overexpress EVI1. Luciferase assays also demonstrated higher activity in Rat1neo cells, indicating higher CAIII expression. Silencing CAIII in Rat1neo cells increased caspase 3 activity after hydrogen peroxide treatment, showing CAIII protects against apoptosis. The results suggest EVI1 overexpression represses CAIII expression, reducing protection against oxidative stress. Therefore, oxidative stress agents may selectively target cancer cells overexpressing
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).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
1. The study analyzed 30 Vibrio cholerae O1 isolates from patients in Delhi, India from 2012-2014 using mismatch amplification mutation assay (MAMA) PCR to identify the ctxB genotype.
2. All isolates were biochemically identified as V. cholerae El Tor serotype Ogawa. MAMA PCR results showed that all isolates carried the ctxB gene of the classical biotype, though they were phenotypically El Tor.
3. This identifies an "altered El Tor" variant circulating in Delhi that displays characteristics of both classical and El Tor biotypes in possessing the classical ctxB gene despite being phenotypically El Tor.
This document summarizes research on the Scr74 gene family in Phytophthora infestans and how different variants of this gene are recognized in potato genotypes. The key points are:
1. 27 variants of the Scr74 effector gene were identified from 12 P. infestans isolates. 4 novel variants were cloned.
2. 13 amino acid sites in Scr74 were found to be under positive diversifying selection, indicating these sites are important for pathogen recognition.
3. 17 Scr74 variants were screened using PVX assays on 12 potato genotypes. Some genotypes had strong cell death responses to specific Scr74 variants, suggesting recognition of these variants.
4. Scr74 variants Scr74-A
This study investigated the role of the cellular protein annexin 2 (Anx2) in human immunodeficiency virus type 1 (HIV-1) particle production and infectivity. The researchers knocked down Anx2 expression by 98% in various cell types using lentiviruses encoding short hairpin RNAs against Anx2. They found that knocking down Anx2 did not reduce HIV-1 virus-like particle production in COS-1, 293T, Jurkat T cells, or primary human macrophages. Similarly, murine cells lacking Anx2 produced normal levels of particles. However, supernatants from Anx2-depleted primary human macrophages exhibited decreased infectivity, indicating Anx2 plays a
The document provides information about midterm exams and extra office hours for a class. It announces that the first midterm exam will be on Thursday, February 15 at 6pm in room 155 Dwinelle, and that review sessions will be held during the regular class time that day. It also notes that the professor will not have office hours on February 13.
Boronated Cetuximab CCR tumor targeting in BNCTkent.riley
This document describes a study evaluating boronated cetuximab (BD-C225) for boron neutron capture therapy (BNCT) of epidermal growth factor receptor (EGFR) positive gliomas. In vitro, BD-C225 showed preferential uptake in EGFR positive glioma cells compared to EGFR negative cells. In vivo, rats with EGFR positive glioma tumors received intracerebral BD-C225, achieving high boron levels in the tumors. BNCT with BD-C225 alone or combined with boronophenylalanine extended survival compared to controls. The results provide support for using molecularly targeted boron delivery agents like BD-C225 for BNCT of brain tumors.
- The document discusses research on the cotton root-knot nematode (Meloidogyne incognita), an endoparasitic nematode that infects thousands of plant species and causes billions in crop damage worldwide.
- The research uses Arabidopsis thaliana plants, including mutant strains, to study the nematode's infection process. Mutant plants have mutations in an ABC transporter gene (At4g15320) involved in secreting chemicals that nematodes use to locate host plants.
- The goal is to see if the mutant transporter gene affects nematode infection rates compared to wild-type Arabidopsis. Results found the mutant was actually not a true mutant and showed similar nematode infection to the
The document summarizes research on expressing HIV-1 antigens in plants as a potential vaccine. The researchers found that transient expression in plants is a viable alternative to transgenic plants. They determined that targeting the HIV-1 Gag protein Pr55Gag and its derivative p17/24 to chloroplasts yielded the highest levels of these proteins. Mice vaccinated with p17/24 produced from chloroplasts developed strong CD4+ and CD8+ T cell responses and HIV-specific antibodies, showing it is a promising vaccine candidate.
This document describes research on cloning and expressing the domain III region of the dengue virus envelope protein (DENV EDIII) from serotypes 3 and 4. Key findings include:
1) DENV EDIII from serotypes 3 and 4 were successfully cloned and expressed in E. coli, and the recombinant proteins were purified using nickel affinity and size exclusion chromatography.
2) The purified recombinant EDIII proteins were able to fold correctly and form stable monomers, as shown through analytical methods like SDS-PAGE and gel filtration chromatography.
3) In mouse studies, immunization with the recombinant EDIII proteins, especially when combined with adjuvants, induced strong humoral and cellular immune responses as measured by
- A single episomal plasmid containing Oct4, Sox2, KLF4, L-Myc driven by a hybrid CAG promoter can reprogram human fibroblasts into induced pluripotent stem cells (iPSCs).
- The inclusion of 5 small molecules that enhance reprogramming results in the appearance of proto-colonies by 7 days and mature colonies by 21 days under low oxygen conditions.
- The iPSCs express pluripotency markers, can differentiate into neurons and cardiomyocytes, and grow robustly in a new xeno-free media. Further work will examine residual vector presence and gene expression profiles.
This study aims to generate fusion-defective mutants of Chlamydomonas reinhardtii through random insertional mutagenesis using glass bead transformation. Transformed cells expressing paromomycin resistance are selected and screened for inability to mate. Several mating-deficient clones have been identified that are defective in swimming, agglutination or both. Further analysis will determine if any clones possess a fusion defect and identify the insertion location to study genes involved in fusion.
This study investigated the role of the transcriptional regulator ToxR in biofilm formation and motility in the pathogenic bacterium Vibrio parahaemolyticus. The study found that:
1) A V. parahaemolyticus mutant lacking ToxR showed decreased biofilm formation and reduced swarming and swimming motility compared to the wild type strain.
2) The ToxR regulator was required for rugose colony formation and expression of genes involved in exopolysaccharide production, which are important for biofilm formation.
3) ToxR did not regulate switching between opaque and translucent colony types in V. parahaemolyticus, which is associated with capsular polysaccharide production.
PRODUCTION OF SEROTYPE 6-DERIVED RECOMBINANT ADENO-ASSOCIATED VIRUS IN SERUM-...Dr. Érica Schulze
This document describes research into producing recombinant adeno-associated virus (rAAV) using serum-free suspension cultures of HEK 293 cells. The researchers investigated the effects of different DNA, polyethyleneimine (PEI) transfection reagent, and cell concentrations on rAAV yields. They found that transfecting at a cell concentration of 3x10^6 cells/mL resulted in a 2.5-3.5 fold increase in infectious virus particles (IVP) and genomic particles (Vg) compared to 1x10^6 cells/mL. When testing different PEI:DNA ratios, IVP ranged from 1.19x10^8 to 1.03x10^9 IVP/
A panel of recombinant monoclonal antibodies against zebrafishShahnaz Yusaf
This document describes the development of 10 recombinant monoclonal antibodies against neural receptors and secreted proteins in zebrafish. The antibodies were generated by expressing the extracellular domains of the target proteins in mammalian cells and using them as antigens. The antibodies were characterized, cloned into expression plasmids, and shown to specifically stain their antigens in fixed zebrafish embryo tissues. The staining patterns matched the known expression patterns of the target genes, demonstrating these antibodies will be useful tools for studying neural development in zebrafish.
Poster - Development of double stand break assessment assay with HCS by using...HCS Pharma
The creation of a double strand break (DSB) is accompanied by the phosphorylation of histone H2AX. The measurement of serine 139 phosphorylated histone H2AX (γH2AX) is reported to be a marker of interest to identify potential genotoxic activity.
In order to evaluate the High Content Screening for γH2AX detection, 4 non genotoxic compounds and 9 genotoxic compounds from the ECVAM list I or II were selected to to be tested on HepG2 cell line. These cells offer the advantage to have H2AX expression data in the literature. In parallel, Human primary keratinocytes were included. Indeed these cells would be relevant for investigating skin adverse effects of topical applied xenobiotics with the advantage of High content imaging as valuable tool for screening in the early discovery phase.
This document describes research into using the recombinant dense granule antigen GRA7 from Toxoplasma gondii for serodiagnosis of toxoplasmosis. The GRA7 gene was amplified from T. gondii genomic DNA and inserted into an expression vector. The recombinant GRA7 protein was expressed in E. coli and purified. Western blot analysis showed human sera reacted with the 29 kDa rGRA7 antigen. ELISA tests using rGRA7 showed sensitivity of 96% for IgM detection and 89% for IgG detection, with specificity of 90% for both, demonstrating its potential usefulness for toxoplasmosis diagnosis.
This Masters thesis defense presentation summarizes research identifying MH class IIβ alleles that may confer resistance or susceptibility to Bacterial Cold Water Disease (BCWD) in rainbow trout. The study genotyped MH class IIβ alleles in six families of trout that had been experimentally infected with the BCWD pathogen. Results found five alleles present among the families. The DAB*1001/DAB*0801 genotype had the lowest hazard ratio, suggesting an association with resistance to BCWD, although results were not statistically significant. Future work could examine MH class I alleles and innate immunity genes to better understand resistance in trout.
2004 paper primer ctx dissemination of ctx m-type -lactamases among clinical ...JairAlexanderTllez
This document summarizes a study analyzing 19 clinical isolates of Enterobacteriaceae (16 E. coli and 3 K. pneumoniae) collected from four hospitals in Paris, France between 2000-2002 that exhibited resistance to extended-spectrum cephalosporins. Testing found the resistance was due to various CTX-M-type beta-lactamase enzymes, predominantly CTX-M-15. Most isolates produced both TEM-1 and CTX-M enzymes. The blaCTX-M genes were located on large plasmids and often upstream of the insertion sequence ISEcp1. Five isolates had identical plasmid fingerprints, suggesting clonal dissemination of CTX-M-15-producing strains in
This document summarizes research investigating the biosynthesis and processing of succinate dehydrogenase (SDH) subunits in cultured mammalian cells. The key points are:
1. Antisera were produced against purified bovine heart SDH and its large and small subunits, which detected precursor and mature forms of the subunits in rat, pig, and bovine cell lines.
2. In pig kidney cells, newly synthesized precursors of the large and small SDH subunits were detected that were 1000-2000 and 4000-5000 Da larger than the mature forms, respectively.
3. Pulse-chase experiments showed the precursor forms were fully processed to the mature subunits within 45 minutes when uncouplers of
This paper describes a regulatory pathway controlling expression of Borrelia burgdorferi OspC and DbpA proteins. The study found that in B. burgdorferi strain 297, the alternative sigma factor RpoN controls expression of the alternative sigma factor RpoS. RpoS then governs expression of the outer surface lipoproteins OspC and DbpA. This regulatory network was determined through targeted gene disruption of rpoN and rpoS, followed by genetic complementation. The findings provide insight into key regulatory networks that impact B. burgdorferi pathogenesis, host range, and virulence.
The ribosomal RNA gene unit of Tritrichomonas foetus was cloned and analyzed. Southern blot analysis showed the rDNA unit is organized as a tandem head to tail repeat of 6 kb, with 12 copies present. The small subunit rRNA is one of the shortest reported at 1571 bp, while the 5.8S rRNA is 159 bp. Northern blot analysis detected primary and precursor rRNA transcripts of 5.8 kb and 4 kb. Sequence analysis confirmed the secondary structure of the small subunit rRNA is similar to other eukaryotes, while being shorter in variable regions.
The document describes the construction of recombinant plasmids containing segments of the rrnB ribosomal RNA operon of E. coli, including the entire operon. Key points:
1) Plasmid pKK 3535 was constructed by cloning the 7.5kb BamHI fragment containing the entire rrnB operon from bacteriophage λ into the BamHI site of plasmid pBR 322.
2) Plasmid pKK 123 was constructed by cloning the 3.2kb EcoRI-BamHI fragment containing part of the 23S rRNA gene into plasmid pBR 313.
3) Plasmid pKK 2361 was constructed by cloning the entire 7.5kb Bam
Molecular Cloning of the Structural Gene for ExopolygalacturonateAlan Brooks
This document summarizes research on the cloning and characterization of a gene (pelX) from Erwinia chrysanthemi that encodes an exopolygalacturonate lyase (ExoPL). The pelX gene was cloned from a mutant strain lacking known pectate lyase genes. ExoPL was purified from a recombinant E. coli strain and characterized. A pelX mutant was constructed in E. chrysanthemi but retained pathogenicity, indicating ExoPL does not contribute to tissue maceration ability.
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Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo
1. 33Gene. 39 (1985) 33-39
Elsevier
GENE 1413
Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo
(Recombinant DNA; DNA mediated gene transfer; expression plasmid; screening; packaging; bacterio-
phage jL)
W. Lindenmaier, K.E.J. Dittmar”, H. Hauser, A. Necker and W. Sebaldb
Departmerzt of Genetics, ‘Cytogenetics and “Invnunobiology, GBF, Gesellschafi fiir Biotechnologische Forschung mbH,
Muscheroder Weg 1, D-3300 Braunschweig (F.R. G.) Tel. (0531)61810
(Received March 28th, 1985)
(Revision received May 2Oth, 1985)
(Accepted June 25th, 1985)
SUMMARY
A method has been developed that allows the isolation of genomic clones from a cosmid library by
homologous recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene.
The genomic cosmid library was packaged in vivo into 1 phage particles. A recombination-proficient host strain
carrying IL2 cDNA sequences in a non-homologous plasmid vector was infected by the packaged cosmid
library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both
vectors were selected. From a recombinant cosmid clone the chromosomal IL2 gene was restored. After DNA
mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively.
INTRODUCTION
IL2, also known as T-cell growth factor, plays a
central role in the cellular immune response. It is a
necessary signal for the proliferation of activated T
lymphocytes and has been implicated in a number of
immunoregulatory functions (for review see Robb,
1984). Human IL2 is a glycoprotein of apparent M,.
of 16 500, and has been purified to homogeneity from
Abbreviations: Ap, ampicillin; bp. base pairs; ConA, concana-
valin A; DMEM, Dulbecco’s-modified Eagle medium; EtBr,
ethidium bromide; FCS, fetal calfserum; HAT, selective medium
containing hypoxanthinc, aminopterin and thymidine (Szybalska
and Szybalski. 1962); IFN. interferon; 1L2, interleukin 2; kb,
1000 bp; Km, kanamycin; PHA, phytohemagglutinine; R. resis-
tance; tk. gene coding for thymidine kinase; TPA, tetradecanoyl
phorbol acetate; u, unit(s); [ 1,designates plasmid-carrier state.
037X-1 Il9~85~$03.30 0 1985 Elscvier Science Publishers
stimulated normal lymphocytes and a human T
leukemia line (Jurkat) (Robb et al., 1984; Conradt
et al., 1985). The coding sequence of the human IL2
gene has been cloned from this cell line and from
lymphoid tissues (Taniguchi et al., 1983, Devos
et al., 1983, Maeda et al., 1983). Recently the iso-
lation of chromosomal DNA fragments containing
the IL2 sequences by conventional screening of
phage i libraries has been described (Fujita et al.,
1983; Degrave et al., 1983; Holbrook et al., 1984).
To study expression and modification of IL2 in
homologous and heterologous animal cells in detail
we decided to isolate the region containing the
chromosomal gene and flanking sequences from a
human cosmid library. For the isolation of specific
cosmid clones we developed a method which uses
the high specificity of homologous recombination
2. i‘l
with the efficiency and c&specificity of the A
packaging system. The development of this simple
genetic screening procedure has been possible after
in vivo packaging of cosmid gene libraries has been
established (Lindenmaier et al., 1982). The proce-
dure is selective and efficient enough to allow the
isolation of single copy genes from a total genomic
cosmid library. Handling of radioactively labelled
probes of high specific activity can be avoided.
MATERIALS AND METHODS
(a) Bacterial strains
Escherichiu coli HB 101 (r ~, m , proA2, Ieu,
supE44, recA 13, rpsL20) and DH 1 (rk ~mk ’ , supE,
gyrA96, recA), kindly provided by H. Lehrach, were
used as recA hosts for hybrid cosmids. E. coli 1400
(hsdS -, met ~, supE, supF, recA56, 3.L512; Cami
and Kourilsky, 1978) was used for in vivo packaging
of the human cosmid library. For recombination
and in vivo packaging of recombinants BHB3169
(W3110r -,m+, i, b2red imm434tsSam7; Poustka
et al., 1984), kindly provided by H. Lehrach, was
used.
(b) Plasmids and cosmids
Cosmid and plasmid DNAs were isolated accord-
ing to the method of Birnboim and Doly (1979).
Vectors pAN26, pV34 (provided by B. Hohn), and
pHC79-2cos/tk (Lindenmaier et al., 1982) were fur-
ther purified by two cycles of CsCl-EtBr centrifu-
gation. pCosIFN/j (Gross et al., 1981) was a gift of
G. Gross. pAN26IFl is described in Table I. The
human cosmid library has been described (Linden-
maier et al., 1984).
(c) Plating cells
Single colonies were streaked on LB-plates and
grown overnight at 37°C (HBlOl and DHl). In vivo
packaging strains were streaked in parallel on two
plates and incubated at 30 aC and 42’ C to verify the
inducibility. NZ-broth containing 0.47; maltose was
inoculated from the fresh overnight plates to an A,,,,,
of about 0.05 and grown with good aeration to
A M,,, = 2.
(d) Animal cell culture and transfection
Mouse Ltk cells were grown in DMEM supple-
mented with lO%FCS. Transfection using the
Ca phosphate precipitation method was carried out
as described (Hauser et al., 1982) employing HAT
selection (Szybalska and Szybalski, 1962). pHIL49
was cotransferred with pHC79-2cos/tk in a molar
ratio of 10 to 1. More than 100 HAT-resistant clones
were combined and grown to confluency. For IL2
production the cells were induced by PHA, ConA,
TPA and calcium ionophore A23187, or mock-
induced and cultivated in RPM1 1640 supplemented
with lOO;, FCS for 20 h.
RESULTS AND DISCUSSION
(a) Recombinant screening of cosmid libraries
The general strategy for gene isolation from cos-
mid libraries by recombination in vivo is shown in
Fig. 1. In principle it is analogous to the method of
Seed (1983) for recombinant screening of Alibraries
(see legend to Fig. 1).
To allow screening by recombination in vivo a pair
of cosmid and plasmid vectors has to be used that
has no homologous sequences. For this purpose a
number of vectors has been constructed that contain
the basic replicon of pBEU 17, a temperature-
inducible copy mutant of plasmid Rl (Uhlin et al.,
1979), and the KmK gene of Tn5 (A. Necker, to be
published elsewhere). These Rl-derived screening
plasmids have no homology to pBR322 and are
compatible with a large number of established
pBR322-derived cosmid vectors (e.g., pHC79,
pJB8) in contrast to the system of Poustka et al.
(1984) which requires use of specific R6-K-derived
cosmid vectors. Cointegrates should only be formed
by recombination via the inserted sequence. Because
formation of cointegrates leads to an increase of
about 4 kb in cosmid size, packaging of the recombi-
nant cosmid can be altered due to the size selection
(38-52 kb; Feiss et al., 1977) exerted by the i,
packaging system.
Cosmid packaging, however, in contrast to i,
growth does not require repeated cycles of infection
and lysis, therefore size selection should be less
3. 35
Cosmld library
ITronsformotlon In VIYO packoglng
Packaged cosmld hbrary
Recombmatlon
IInfectlon+selectlon of ApR-KmPrecomb~nants
4, Reversion of recomblnatlon
Fig. I. Recombinant screening method using in vivo packaged
cosmid libraries. (1)The sequence of interest (IL2) is cloned in
pAN26, a small screening vector, and used to transform a recA +
E. coli host strain carrying a temperature-inducible i prophage
(it’ = &wn434ts). By heat induction the cosmid library is pack-
aged in viva forming transducing particles. (2) After transduction
of the library into the screening strain recombination between
homologous sequences can occur. A recombination event leads
to the formation of cointegrates that carry the antibiotic resist-
ance markers of both vectors in cis. (3) Recombinants are
separated from nonrecombinants by in viva packaging, trans-
duction in a new host and selection of ApRKmR colonies. (4) The
genomic structure can be restored by reversion of the recombi-
nation event.
pronounced. For very large cosmids the recombi-
nation products might be packaged with decreased
efficiency.
To test the specificity and efficiency of the recom-
bination screening method, a small Hind111 fragment
(fragment G) of a cosmid containing the human
IFNP chromosomal gene cloned in pJB8 (Gross
et al., 198 1) was subcloned using pAN26 (Fig. 2) in
E. coli BHB3169. pCosIFNb was transformed into
E. cofi 1400 and packaged in vivo. The recombi-
nant strains BHB3169[pAN26] and BHB3169-
[pAN26IFl] were infected with cosIFNP lysate,
ILimcDNA ,n pHUMIL2
2s Rs’Hf X St Hi P5
5 .- 3
Fig. 2. Construction of the screenmg plasmid pAN261L2. Sites
for restriction enzymes are indicated as Ps, PsrI; Hf, HintI;
X. Xbal; St, Stul; H, HindIII; Bg, &/II: S, &z/I; Rs’, f&I site
created by connecting the G-tail to nucleotide 113 of IL2-cDNA
(Taniguchi et al., 1983; W. S., unpublished data). IL2-coding
region: dark-shaded bar; 3’ noncoding region: black bar; Rl
replicon: open bar; Kmn region from Tn5: hatched bar.
grown and induced for in vivo packaging as de-
scribed in the legend to Table I. After infection of
HBlOl and selection for KmR and ApR clones no
colonies were found using BHB3 169[pAN26],
whereas doubly resistant recombinants between
cosIFN/ and pAN26IFl could be isolated with a
frequency of about 1 x 10 4 (Table I). Restriction
analysis of recombinant cosmids showed the expect-
ed structure (not shown).
For the isolation of the chromosomal IL2 gene
from a human cosmid library by recombinant
screening, human IL2 cDNA sequences cloned in
pBR322 (W. S., unpublished data) were inserted into
pAN26. The central 450-bp Hi&I fragment of the
pHUMIL2 cDNA insert was subcloned into pAN26
(Fig. 2). The resulting plasmid, pAN26IL2 was
transformed into E. coli BHB3169 for recombi-
nation screening. The approx. 300000 primary
clones of the human cosmid library in E. coli 1400
were amplified as single colonies, resuspended and
induced for in vivo packaging (Lindenmaier et al.,
1982; 1984). The packaged library can efficiently be
transferred to a host providing recombination and
packaging functions for a limited period of time.
About 10’ packaged cosmids were used to infect
4. 36
TABLE I
Efficiencies of in viva recombination
Screening plasmid
in BHB3169
pAN26
pAN261L2
pAN26IFl’
pAN26
pAN261L2
Cosmid lysate”
used for
infection
pCosIFNfi
pCoslFNp
pCosIFN/i
cosmid library
cosmid library
Infected cells
before addition
of antibiotics
2.3 x IO’
1.8 x IO”
2.9 x IO’
1.3 x lOX
3.7 x 10%
Titer of Titer of Recombination
packaged cosmidsh packaged cosmids” frequency
(Apn trans- (ApKKmR
ductants/ml) transductants/ml)
2x lox 0 <5 x 10 <’
3x IO’ 0 <3x10 x
1.0 x lox 10 000 Ix10 J
9x IOX 70 7.x x 10 s
3.5 x lox 45 1.3 x IO ’
” In viva packaging of cosmid pCosIFNp (Gross et al., 1981) and the human cosmid library were done essentially as described
(Vollenwcider et al., 1980; Lindenmaier et al., 1982).
’ For recombinant screening, cells of BHB3169 harbouring the screened plasmid were prepared and infected with cosmid lysate by
incubation at 30°C 20 min. Infected cultures were diluted tenfold with L-broth and incubated for 40 min at 30°C. Infected cells
were selected by adding Km and Ap to 15 pg;mI each. Incubation at 3O’C was continued for 2 h more at 3O’C. .4fter induction and
in vwo packaging, induced cells were pelleted by centrifugation, resuspended in l/20 vol. of TM buffer (50 mM Tris HCI pH 7.5.
10 mM MgCI,) and lysed by addition of chloroform. Cell debris was pelleted by centrifugation and the supernatant used as cosmid
lysate. HBlOl plating cells were infected to determine the titer of ApR transductants and ApRKnR recombinants.
i pAN261Fl was constructed by inserting HirzdIII fragment G (0.55 kb) of pCosIFNP into the unique HiftdIII site of pAN26.
0
5 E. coli BHB3 169[ pAN26IL21. After recombination
and repackaging, packaged cosmids were used to
infect E. coli HBlOl. ApRKmK colonies were found
with a frequency of about 1 x 10 ~‘. In control expe-
riments using BHB3169[pAN26] in combination
with the human genomic library ApKKmR colonies
appeared with about the same frequency (Table I).
We do not know whether this is due to a type of
transient recombination as described for Ml3
phages (Dagert and Ehrlich, 1983) or to the presence
m of sequences homologous to pAN26 in the DNA
L/
library. But in spite of this nonspecific background,
true recombinants should be highly enriched in the
doubly resistant colonies.
From the recombination screening experiment
with pAN26IL2 we analyzed 24 ApRKmK colonies
by digestion of cosmid DNA with BglII and PvuII
and Southern blotting. Two of the clones contained
fragments hybridizing to IL2 cDNA different from
the internal fragment of pAN26IL2. One of them
(cosHIL24) contained three BglII fragments hybrid-
izing to IL2 cDNA and was analyzed in more detail
(Fig. 3). Hybridization to pAN26IL2 showed that it
Fig. 3. Southern hybridization of BglII-digested DNA of recom- pHUMIL2 (2), pAN261L2 (3). Hinfl-PstI fragment (4) and
binant cosmid cosHIL24. EtBr-stained agarose gel (I) and RwI-HinfI fragment (5) of the pHUMIL2 insert (see Fig. 2).
autoradiograms of hybridization to IL2-cDNA insert of Sizes in kb are on the left margin.
5. 37
contained the recombinant plasmid integrated into
the cosmid DNA. A Z&I-Hi&I fragment repre-
senting 5’ sequences and the HinfI-PstI fragment
corresponding to 3’-untranslated sequences were
isolated from pHUMIL2. These sequences are not
contained in the recombinant plasmid pAN26IL2.
When the 5’ and 3’ specific probe was hybridized to
BgZII-digested cosHIL24 DNA a 9.5kb and a
6.5kb fragment, respectively, hybridized specifi-
cally. This means that a cosmid clone containing
human genomic IL2 sequences extending into the 5’
and 3’ direction from the recombinant sequence has
been isolated by specific recombination.
(b) Restoration of the genomic IL2 gene structure
In the recombinant cosmid cosHIL24 the se-
quence of the gene is interrupted by the recombinant
plasmid pAN26IL2. These sequences have to be
removed to restore the genomic structure. In prin-
ciple this can be achieved by reversion of the recom-
bination event in vivo. The intramolecular recombi-
nation leading to excision of the recombinant plas-
mid spontaneously occurs in vivo with low efficiency
even in recA - host strains, as can be seen by the
appearance of the small recombinant plasmid. It can
be enhanced by providing the functions for homol-
ogous recombination, e.g., by transfer into recA +
E. coli strains or by superinfection with hed’ phage
(Poustka et al., 1984; our unpublished results). This
second step probably does not occur at exactly the
same position as the first one and could result in the
formation of hybrid genes. In the case of cosHIL24
direct reversion was not possible. RecA + conditions
resulted in a number of large deletions not confined
to pAN26IL2. This instability of cosHIL24 is not a
general feature of cointegrates. Other isolates were of
normal stability.
To restore the chromosomal structure of the IL2
gene we had to use in vitro recombination. The IL2-
coding sequence contains a single XbaI site
(Taniguchi et al., 1983). In cosHIL24 the integration
of pAN26IL2 occurred in the third exon, which
contains the corresponding XbaI site leading to a
duplication of this site. Removal of this XbaI frag-
ment should restore the original structure. To do so
the BgrII fragments containing IL2 coding sequences
have been subcloned into the BglII site of pV34 (Fig.
4). Restriction maps of the subclones confirmed the
expected structure. Restoration of the genomic IL2
gene was achieved in two cloning steps (Fig. 4). The
resulting plasmid (pHIL49) represents 12.8 kb of the
genomic region. It was mapped with a number of
restriction enzymes and showed the structure ex-
pected from the mapping of cosHIL24 and the sub-
clones, except that pAN26IL2 sequences are deleted
(Fig. 4B). It is also in agreement with restriction
maps derived from published sequences of part of
this region, except for one XbaI site (Fujita et al.,
1983, Degrave et al., 1983, Holbrook et al., 1984).
There are no discrepancies between fragments of
human placental DNA digested with EcoRI,
HindIII, BgZII and PvuII hybridizing to IL2 cDNA
(not shown) and the fragments contained in pHIL49.
(c) Expression of human IL2 in mouse L-cells
To show that the human IL2 gene in pHIL49 is
functional, it was introduced into mouse Ltk cells
TABLE II
IL2 activity in supernatants of Lrk- mouse cells transfected with
pHlL49 containing the restored human IL2 gene
Medium IL2 activity Control Medium
(u/ml)” cells control
Transfected cells
Exp. 1 Exp. 2
RPM1 1640 0.11 0.4 <O.Ol <O.OI
RPM1 1640
+lO;‘, FCS 0.13 0.25 <O.Ol < 0.01
RPM1 1640
+20 pg/ml PHA 0.12 0.21 nt <O.Ol
RPM1 1640
+lO pg/ml ConA 0.12 0.12 <O.Ol <O.Ol
RPM1 1640
+lO pg/ml TPA
+I0 pg/ml A23187 0.13 0.1 GO.01 <O.Ol
il IL2 activity was determined as the T cell growth factor-
mediated [3H]thymidine incorporation into the strictly IL2 de-
pendent murine T cell line CTLL-2, which was kindly provided
by Dr. H.H. Peter (Freiburg, F.R.G.). Briefly, IL2-containing
supernatants were serially diluted on 96-well culture plates in
RPM1 1640 medium supplement with 10% heat-inactivated FCS.
4 x lo3 cells were plated per well and incubated for 24 h at 37°C.
After an [3H]thymidine pulse (1 pCi/well; I Ci/mmol) for 4 h the
cells were harvested with an automatic cell harvester. The IL2
activity was expressed as u/ml.
6. 38
a pv 3L
P
0
89
BS
Pvu II
Born HI
PHIL ZL-31
P
B
S
a
Bg
S
89
P
P
“I x
PHIL 268
P
PHIL-ZL-3
PHIL L9 P
b
Fig. 4. Restoration of the chromosomal IL2 gene structure. (a)
Subclones pHIL24-43, pHIL24-31 and pHIL24-3, containing
the 9.5-kb, 3.1-kb and 6.5-kb Bg111 fragments of cosHlL24,
were digested with the enzymes indicated and the fragments
corresponding to genomic IL2 sequences isolated. In the tirst
step the PwII-XhaI fragment of pHIL24-43 and the partial
XhaI + BumHI fragment of pHIL24-31 were cloned into
PvuII + BnmHI-digested pV34 to remove the recombinant plas-
mid sequences. The resulting plasmid pHIL24-8 was digested by
EJ$II +&II, and the 3’ fragment isolated from pHIL24-3 by
digestion with the same enzymes was inserted. Open bars:
human genomic sequences; hatched bars: pAN26 sequences. (b)
Restriction map of the restored region of the human IL2 gene
contained in pHIL49. Sites for B, BarnHI; Bg, &$II; C, &I;
E, EcoRI; H, HirzdIII; P, PvtrII: S, &if; St, SflrI and X, XbnI
arc indicated. Black bars indicate coding regions.
using the cotransfer technique (Wigler et al., 1979).
Stable HAT-resistant clones were selected and su-
pernatants tested for IL2 activity on the strictly IL2-
dependent mouse cell line CTLL2. Supernatants of
clones stably transfected with pHIL49 showed low
but clearly detectable levels of constitutive expres-
sion of IL2 activity (Table II). When mouse L cells
transfected with the human IL2 gene were treated
with PHA, ConA, TPA or TPA plus calcium iono-
phore A23 187, agents that induce IL2 production in
peripheral blood lymphocytes and Jurkat cells
(Robb, 1984), no increase in the amount of IL2 pro-
duced could be detected (Table II). This lack of
inducibility could be due to the absence of regulatory
DNA sequences on pHIL49 controlling the induc-
tion and/or the lack of other regulatory factors from
the host cells which might be species- and/or differ-
entiation-specific.
The IL2 activity produced by mouse L cells trans-
fected with pHIL49 gene was further characterized
by gel filtration. It eluted at exactly the same position
IL
u/n
10
i0 i5 50 ml
elutlon volume
Fig. 5. Gel filtration on Sephadex G75. IL2 activities produced
by L& mouse cells transfected with pHIL49 (0) were
compared to mouse IL2 (*) and human IL2 (0). M, markers
were dextran blue (I), bovine serum albumin (2), ~~valbumin (3),
myoglobin (4) and phenol red (5). Serum-free culture superna-
tants wcrc concentrated by (NH&SO, precipitation (80”” w/w).
The pellet was dissolved in 10 mM Hepes pH 7.4, 100 mM NaCl
and fractionated on Sephadex G-75 column. The elated fractions
were collected over FCS (final ~~)ncentrati~)n IO”,, VT). filter-
sterilized and tested for IL2 activity.
7. 39
as authentic human IL2 from Jurkat cells or normal
human lymphocytes and was clearly distinguishable
from mouse IL2 (Fig. 5). To see whether posttrans-
lational processing and modification is identical to
authentic human IL2, which is ~-glycosylated at
threonine in position 3 (Robb et al., 1984, Conradt
et al., 1985), more efficient production, purification
and analysis will be required.
ACKNOWLEDGEMENTS
WC thank Dr. J. Collins for support and helpfut
discussions. We also thank R. Bonewald and H.
Schliephake for expert technical assistance and B.
Seeger-Kunth for typing the manuscript. This work
was supported by a grant from the Bundesministe-
rium fiir Forschung und Technologie.
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