Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “cloning”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources
PubMed1995

A transformed murine myocardial vascular endothelial cell clone: characterization of cells in vitro and of tumours derived from clone in situ.

In the course of maintaining a cloned murine myocardium-derived endothelial cell line (mouse heart endothelial cell clone 5; MHEC5) a spontaneously transformed variant has been identified (clone MHEC5-T). On injection into histocompatible mice, clone MHEC5-T uniformly generated epithelioid haemangioendotheliomas. Clone MHEC5-T underwent significant additional alterations in addition to the acquisition of tumour-forming potential in vivo along with the diagnostic correlate of loss of cellular contact inhibition in vitro. Whereas the transformed cells maintained lectin-binding properties characteristic of endothelial cells, they lost the cell surface receptor(s) for acetylated low density lipoprotein and no longer bound antibodies to either angiotensin converting enzyme or von Willebrand factor-associated antigen. Vascular cell adhesion molecule-1 (VCAM-1), expressed constitutively on the parent clone, was down-regulated in the transformed cell line. The transformed cells acquired immunoreactivity to antibodies directed against cytokeratin, and they showed a markedly increased response to migration-inducing factors in vitro. The cell line described in this report demonstrates that the in vitro transformation of myocardium-derived endothelial cells can lead through transitional stages of differentiation to a new stable phenotype characterized by endothelial--to--epithelioid transition. The study of MHEC5-T cells, in addition to providing insight into the biology of cardiac neoplasms, may help to elucidate regulatory mechanisms involved in endothelial cell activation, transition and transformation.

Animals↗
PubMed1984

Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis. I. Construction and analysis of B. subtilis clone banks in Escherichia coli.

Cloning in Escherichia coli and Bacillus subtilis was carried out using the bifunctional plasmid pDH5060. B. subtilis chromosomal DNA and pDH5060 DNA were digested with either BamHI or SalI, then annealed, ligated, and transformed into E. coli SK2267. Transformants containing sequences ligated into the BamHI or SalI sites in the Tcr gene of pDH5060 were selected directly using a modification of the fusaric acid technique. The BamHI and SalI clone banks contain about 250 and 140 B. subtilis fragments, respectively, with an average insert size of 8-9 Kbp in the BamHI and 4-5 Kbp in the SalI bank. The inserts ranged in size from 0.3 Kbp to greater than 20 Kbp. The vector used here therefore accepts inserts which are significantly larger than previously reported for other B. subtilis cloning systems. All individual cloned B. subtilis sequences examined were stably propagated in E. coli SK2267. Eight of eighteen B. subtilis auxotrophic markers tested (aroG, gltA, glyB, ilvA, metC, purA, pyrD, and thrA) were transformed to prototrophy with BamHI or SalI clone bank DNA. All or part of the hybrid plasmid DNA recombined at the sites of homology in the chromosome of these Rec+ recipients. Loss of sequences from hybrid plasmids was not prevented in a r- m- recE4 recipient strain of B. subtilis. Although the recE4 background prevented recombination between homologous chromosomal DNA, a variety of cloned fragments were shown to be unstable and undergo deletions of both insert and plasmid sequences. In addition, B. subtilis sequences propagated in E. coli transformed B. subtilis recE4 recipients with a 500-1,000-fold reduced efficiency.

Bacillus subtilis↗
PubMed1989

Isolation of encephalitogenic CD4+ T cell clones in the rat. Cloning methodology and interferon-gamma secretion.

In this report we detail a procedure for the cloning of a rat encephalitogenic T cell line and show that the methods normally employed for other species may not always be applicable. The two important differences to be described are, (i) that in these experiments where the parent T cell lines were generated with thymocytes as presenting cells, splenocytes were not suitable as a source of antigen-presenting or stimulator cells and (ii) semipurified forms of IL-2, specifically that derived from EL4 lymphoma cells, resulted in a much reduced cloning frequency and rate of T cell growth compared with cruder mixtures such as that derived from mitogen-stimulated splenocytes. Functional studies with clones derived from a strongly encephalitogenic (experimental autoimmune encephalomyelitis (EAE)-inducing) T cell line revealed that the clones had a reduced capacity to mediate EAE in recipient rats but were otherwise comparable to the parent line in terms of surface phenotype and fine antigen specificity. In an attempt to begin to identify the type of CD4+ T cells that may induce EAE we tested the clones and lines for secreted interferon-gamma by a sensitive ELISA, and showed that all clones secreted high levels of this factor.

Animals↗
PubMed1993

Effects of media conditioned by a non-metastasizing human salivary gland adenocarcinoma cell clone and metastasizing clones from salivary gland and various other tissues on the proliferation, migration and protease production of bovine aortic endothelial cells in vitro.

We demonstrate the role in tumor-associated angiogenesis of factors released into conditioned medium (CM) from in vitro human salivary gland cell clones with biological phenotypes ranging from non-metastasizing to metastasizing. A non-metastasizing human salivary gland adenocarcinoma cell clone HSGc and its subclone with metastatic potential (Gc2-100 cl-1) were employed. We also used metastasizing cell clones obtained by explant cultures of organs of Gc2-100 cl-1 tumor-bearing nude mouse. The proliferation and migration of bovine aortic endothelial (BAE) cells were significantly stimulated by the addition of CM obtained from Gc2-100 cl-1 and metastasizing cell clones, while CM from HSGc was ineffective. When the effect on protease secretion by BAE cells was examined, CM from Gc2-100 cl-1 and metastasizing cell clones inhibited the secretion of type IV collagenases by BAE cells much more than did CM from HSGc. These findings, therefore, may imply that Gc2-100 cl-1 and metastasizing cell clones secrete angiogenic factors that stimulate not only the proliferation and migration of endothelial cells but also the formation of basement membrane components necessary for the reconstruction of new blood vessels at migrated sites of endothelial cells by preventing the degradation of basement membrane component, type IV collagen.

Adenocarcinoma↗
PubMed2006

Using a modified TA cloning method to create entry clones.

We describe a noncommercial alternative method to create entry clones compatible with all kinds of destination vectors based on an improved TA cloning approach. To generate Gateway T vectors, we first constructed gentamicin- and chloramphenicol-resistant entry vectors designated pGWG and pGWC, respectively. Each entry vector contains an AhdI cassette flanked by attL sites, with each AhdI cassette containing two AhdI restriction enzyme sites spaced by the ccdB killer gene, which is lethal to most Escherichia coli strains. Gateway T vectors can be prepared by simple digestion of these entry vectors with the AhdI enzyme or its isoschizomers. The use of the ccdB gene as a negative selection marker is an important improvement over conventional TA cloning in that it eliminates the necessity of blue/white color screening based on alpha-complementation. Another important improvement that we have implemented is to retail the T vectors using Taq polymerase and dTTP so as to improve the cloning efficiency. Together, these improvements allow TA cloning to realize its full potential. Using Gateway T vectors prepared by this improved method, entry clones for PCR products or restriction enzyme fragments can be created simply, efficiently, and inexpensively while at the same time introducing greater compatibility.

Chloramphenicol↗
PubMed2006

A comparative analysis of shotgun-cloning and tagged-random amplification-cloning of chromatin immunoprecipitation-isolated genome fragments.

The cloning of transcription factor antibody-immunoprecipitated genomic fragments from chromatin immunoprecipitation (ChIP) experiments is a technically challenging procedure, especially when the input genomic DNA is isolated from whole tissues (in vivo) rather than cultured cells. Here we adapt a technique known as Tagged-Random PCR (T-PCR) to amplify ChIP-immunoprecipitated DNA from mouse embryonic tissue prior to cloning. Importantly, we then compare this technique with tandem shotgun-cloning experiments in terms of its capacity to identify target genes. We find that T-PCR dramatically increases the efficiency of cloning ChIP fragments without distortion of the relative location of cloned fragments to putative target genes. Thus, T-PCR is a simple procedure which greatly enhances the efficiency of cloning tissue-derived ChIP fragments.

Animals↗
PubMed2005

High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system.

The success of structural genomics and proteomics initiatives is dependent on the availability of target genes in vectors suitable for protein production. Here, we compare two high-throughput methods for producing expression vectors from plasmid-derived cDNA fragments. Expression vectors were constructed for compatibility with the Gateway recombination cloning system and the Flexi Vector restriction-based cloning system. Cloning protocols for each system were conducted in parallel for 96 different target genes from PCR through the production of sequence-verified expression clones. The short nucleotide sequences required to prepare the target open reading frames for Flexi Vector cloning allowed a single-step PCR protocol, resulting in fewer mutations relative to the Gateway protocol. Furthermore, through initial cloning of the target open reading frames directly into an expression vector, the Flexi Vector system gave time and cost savings compared to the protocol required for the Gateway system. Within the Flexi Vector system, genes were transferred between four different expression vectors. The efficiency of gene transfer between Flexi Vectors depended on including a region of sequence identity adjacent to one of the restriction sites. With the proper construction in the flanking sequence of the vector, gene transfer efficiencies of 95-98% were demonstrated.

Cloning, Molecular↗
PubMed1981

Antigen-specific human T-cell clones: development of clones requiring HLA-DR-compatible presenting cells for stimulation in presence of antigen.

A method for cloning soluble antigen-specific proliferating human T lymphocytes directly from peripheral blood cells of individuals recently primed with the antigen is described. The soluble antigen was keyhole limpet hemocyanin. After expansion in liquid culture, these cells were shown to be antigen specific and to require HLA-DR-histocompatible presenting cells. Recloning of small numbers of these cells in soft agar under conditions of high plating efficiency yielded true clones (i.e., derived from a single cell) which retained specificity for the original stimulating antigen as shown by blastogenic responses measured by incorporation of [3H]thymidine. However, antigen-specific stimulation of clones was demonstrated only in the presence of an HLA-DR-compatible cooperating cell population together with the relevant antigen. Cells that manifested only HLA-A or B locus identity with the T-cell clone were not effective as presenting cells. Other antigens, such as tetanus toxoid, to which the donor of the clone or of the presenting cells was immune failed to stimulate the clone.

Antigens, Surface↗
PubMed1982

Open reading frame cloning: identification, cloning, and expression of open reading frame DNA.

A plasmid was constructed that facilitates the cloning and expression of open reading frame DNA. A DNA fragment containing a bacterial promoter and the amino terminus of the cI gene of bacteriophage lambda was fused to an amino-terminally deleted version of the lacZ gene. An appropriate cloning site was inserted between these two fragments such that a frameshift mutation was introduced upstream of the lacZ-encoding DNA. This cloning vehicle produces a relatively low level of beta-galactosidase activity when introduced into Escherichia coli. The insertion of foreign DNA at the cloning site can reverse the frameshift mutation and generate plasmids that produce a relatively high level of beta-galactosidase activity. A large fraction of these plasmids produce a fusion protein that has a portion of the lambda cI protein at the amino terminus, the foreign protein segment in the middle, and the lacZ polypeptide at the carboxyl terminus. The production of a high level of beta-galactosidase and a large fusion polypeptide guarantees the cloning of a DNA fragment with at least one open reading frame that traverses the entirety of the fragment. Hence, the method can identify, clone, and express (as part of a larger fusion polypeptide) open reading frame DNA from among a large collection of DNA fragments.

Base Sequence↗
PubMed1981

T cell clones reactive with sperm whale myoglobin. Isolation of clones with specificity for individual determinants on myoglobin.

We have been able to isolate clones of sperm whale muscle myoglobin (Mb)-reactive T cells from (C57BL/6 x A/J)F1 [(B6A)F1] mice. Four types of clones were isolated, distinguished by their patterns of recognition of Mb cyanogen bromide (CNBr) fragments and antigen presenting cell (APC) requirements. Individual T cell clones proliferated in response to one of three CNBr fragments of Mb. Dose-response curves of all clones were identical for native Mb and the appropriate fragment. T cell clones reactive to fragment 1-55 did not proliferate in response to peptide 15-22 (a peptide that binds to serum antibody directed against 1-55). These data support previous findings suggesting differences between antigen recognition by T and B cells, i.e., T cells may not recognize antigen in its native conformation and/or T and B cells may recognize distinct epitopes on the same antigen. Using T cell clones to analyze genetic control of responsiveness to Mb, we found that certain (B6A)F1 T cells recognize Mb presented by low responder strain APC. Thus, genetically determined low responsiveness in this case is probably not due to failure of APC function. We also found that responsiveness to certain Mb epitopes mapped to the I-A subregion whereas others mapped, via gene complementation, to the I-A and I-E subregions. We found no examples of responsiveness mapping to the I-C subregion and suggest an alternative explanation for previous reports mapping genetic control of responsiveness to certain Mb determinants to I-C.

Animals↗
PubMed1983

Analysis of cross-reactive antigen-specific T cell clones. Specific recognition of two major histocompatibility complex (MHC) and two non-MHC antigens by a single clone.

Two T cell clones, one specific for I-Es/d plus myelin basic protein (BP) and another specific for I-Ak plus influenza virus have been demonstrated to cross-react with DBA/2 cells. Genetic and serological analyses have shown that each clone recognizes its respective priming antigen in association with self-major histocompatibility complex (MHC) determinants and each recognizes DBA/2 minor H antigens in association with allo I-Ad MHC antigens. Further analysis of these clones suggests (a) that the allo I-Ad MHC epitopes recognized by these clones are not shared with self-I-A epitopes, (b) that the virus or BP antigens do not cross-react with DBA/2 minor H antigens, (c) that these clones recognize different determinants on the DBA/2 minor H antigens, and (d) that there is a requirement for a specific association between the different MHC antigens and the non-MHC antigens to stimulate these clones. This specific associative recognition argues strongly for the "altered self" hypothesis.

Animals↗
PubMed1986

Heterogeneity of clones from a human metastatic melanoma detected by autologous cytotoxic T lymphocyte clones.

The possibility that a single human tumor may be composed of an heterogeneous population of cells with respect to susceptibility to lysis by autologous CTL clones was investigated by testing six cytolytic clones derived by micromanipulation against the autologous metastatic melanoma, Me28, and against 31 clones derived from Me28 by cloning in soft agar. Highly significant differences in the lysis of many tumor clones were observed by three of the CTL effectors in comparison with the cytotoxicity achieved on Me28. These results indicate that cloned cellular reagents can detect heterogeneity among cells isolated from the same melanoma, and suggest that the target determinants recognized on the autologous tumor might be differentially expressed on different neoplastic cells.

Analysis of Variance↗
PubMed2005

Preparation of a set of expression-ready clones of mammalian long cDNAs encoding large proteins by the ORF trap cloning method.

Although we have so far identified and sequenced >2000 human long cDNAs, known as KIAA cDNAs, half of them have yet to be functionally annotated. Expression-ready cDNA clones derived from these genes, where the open reading frame (ORF) of the gene of interest is placed under the control of an appropriate promoter, are critical for functional characterization of these gene products. In this study, we attempted to systematically convert original cDNA clones to expression-ready forms for native and fusion proteins. For this purpose, we developed a new method for ORF cloning based on a homologous recombination in Escherichia coli to avoid laborious manipulations and artificial introduction of mutations in ORF. Using 1589 putative full-length ORFs (from 1002 KIAA genes, 119 human known genes and 468 mouse genes) with an average size of 2.8 kb, we successfully prepared expression plasmids for 1463 native proteins and for 1343 fusion proteins by this method. The resultant expression-ready clones were examined using an in vitro transcription/translation system followed by SDS-polyacrylamide gel electrophoresis and by transient expression of GFP-fusion proteins in human embryonic kidney (HEK) 293 cells. This set of expression-ready clones of long cDNAs encoding large proteins would open a new route to experimentally analyze their functions on a proteomic scale, since unavailability of expression-ready clones for mammalian large proteins has been a major obstacle to the functional analysis of these cDNAs.

Animals↗
PubMed1996

Cloning and characterization of the major histone H2A genes completes the cloning and sequencing of known histone genes of Tetrahymena thermophila.

A truncated cDNA clone encoding Tetrahymena thermophila histone H2A2 was isolated using synthetic degenerate oligonucleotide probes derived from H2A protein sequences of Tetrahymena pyriformis. The cDNA clone was used as a homologous probe to isolate a truncated genomic clone encoding H2A1. The remaining regions of the genes for H2A1 (HTA1) and H2A2 (HTA2) were then isolated using inverse PCR on circularized genomic DNA fragments. These partial clones were assembled into intact HTA1 and HTA2 clones. Nucleotide sequences of the two genes were highly homologous within the coding region but not in the noncoding regions. Comparison of the deduced amino acid sequences with protein sequences of T. pyriformis H2As showed only two and three differences respectively, in a total of 137 amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes arose before the divergence of these two species. The HTA2 gene contains a TAA triplet within the coding region, encoding a glutamine residue. In contrast with the T. thermophila HHO and HTA3 genes, no introns were identified within the two genes. The 5'- and 3'-ends of the histone H2A mRNAs; were determined by RNase protection and by PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly expressed in vegetatively growing cells but only weakly expressed in starved cultures. With the inclusion of these two genes, T. thermophila is the first organism whose entire complement of known core and linker histones, including replication-dependent and basal variants, has been cloned and sequenced.

Amino Acid Sequence↗
PubMed1989

Polyclonal rabbit gamma globulins against a human cytotoxic CD4+ T cell clone. I. Clone characteristics and antiblast globulin preparation.

Antihuman lymphocyte rabbit (or horse) gamma globulins used in recipients of organ transplantation are prepared against thymocytes or immortalized cell lines, the only two sources so far allowing enough antigen preparation. These cells lack, however, the surface determinants characteristic of alloreactive blasts involved in the rejection process. We have derived long-term cultures of a panel of alloreactive (untransformed) clones from a rejected kidney. Among them, clone 1E7 has been chosen as a cytotoxic CD4+ (CD2+ CD3+ TCR alpha beta+) clone proliferating against HLA-DR8 targets. This clone (clonality assessed on T cell receptor genomic rearrangements) has been grown using weekly stimulations with the kidney donor-derived EBV cell line and recombinant IL-2. Clone cultures have been adapted to mass production after optimization of culture conditions satisfying pharmaceutical requirements. This procedure warranted a reproducible source of antigen since the functional and phenotypic characteristics of the immunizing 1E7 cells remained identical through the life span of the culture. In addition, the study of the total growth capacity of 1E7 cells showed consistent expansion until the 40th cell cycle, ensuring a progeny that will satisfy the large-scale requirement for a clinical trial. Rabbits were injected with 100 x 10(6) 1E7 cells (21, 14, and 7 days before bleeding). Sera were depleted of agglutinin by red blood cell absorption and globulin antiblast (GAB) prepared by SO4Na precipitation and ion exchange chromatography; 50% complement-mediated target cell lysis and 50% inhibition of E rosette formation and alloproliferation were obtained at GAB dilutions of about 1:250-1:500. Prescreened on cynomolgus monkeys, GAB could significantly prolong skin grafts when given prophylactically. Finally GAB have been used in human recipients of kidney grafts for prophylaxis of early rejection. Results of this pilot study are given in a separate report in this issue. In conclusion we have, for the first time, set up a large-scale preparation of polyclonal globulin against a normal human alloreactive clone, and this new reagent should present several advantages over classic antilymphocyte or antithymocyte sera because it contains specificities against activation antigens and has less crossreactivity variation among batches.

Animals↗
PubMed1989

Cloning and characterization of Treponema hyodysenteriae antigens and protection in a CF-1 mouse model by immunization with a cloned endoflagellar antigen.

We cloned genes that code for Treponema hyodysenteriae antigens into Escherichia coli with the purpose of identifying protective antigens for vaccine development. Three different genomic libraries were screened with various antisera reactive with T. hyodysenteriae antigens. The cloned antigens and corresponding native T. hyodysenteriae antigens were analyzed for molecular size, serum reactivity, solubility in sarcosine, and segregation during phase partitioning with the nonionic detergent Triton X-114. The results from these analyses suggested that the gene products were components of either the cytoplasmic membrane, periplasm, or endoflagella of T. hyodysenteriae. The cloned antigens were tested as vaccine candidates in a CF-1 mouse model of T. hyodysenteriae infection and immunity. Intraperitoneal injection of crude E. coli extracts containing cloned antigens did not protect mice from challenge. However, serum from mice injected with a crude extract of an E. coli clone which expressed an endoflagellar antigen killed T. hyodysenteriae in vitro. Partially purified preparations of this cloned endoflagellar antigen protected mice against oral challenge with both the homologous serotype (B204) and a heterologous serotype (B234) of T. hyodysenteriae. These results suggest that the endoflagellar proteins could be used as an effective subunit vaccine against T. hyodysenteriae.

Animals↗
PubMed1999

To clone or not to clone--a Jewish perspective.

Many new reproductive methods such as artificial insemination, in vitro fertilisation, freezing of human embryos, and surrogate motherhood were at first widely condemned but are now seen in Western society as not just ethically and morally acceptable, but beneficial in that they allow otherwise infertile couples to have children. The idea of human cloning was also quickly condemned but debate is now emerging. This article examines cloning from a Jewish perspective and finds evidence to support the view that there is nothing inherently wrong with the idea of human cloning. A hypothesis is also advanced suggesting that even if a body was cloned, the brain, which is the essence of humanity, would remain unique. This author suggests that the debate should be changed from "Is cloning wrong?" to "When is cloning wrong?".

Cloning, Organism↗
PubMedDate not supplied

Comparative analysis by polymerase chain reaction amplified minicircles of kinetoplast DNA of a stable strain of Trypanosoma cruzi from São Felipe, Bahia, its clones and subclones: possibility of predominance of a principal clone in this area.

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of São Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicircles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment length polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%. This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.

Animals↗