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Query: UNIPROT:P06889 (Mol)
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A new retrovirus vector containing the gene for hygromycin B resistance (hyg) as a selectable marker under the control of an internal simian virus 40 promoter was constructed. It was used, together with an analogous previously described vector, DO1, which contains the gene for G418 resistance, to introduce and express the genes for the two chains of a human class II major histocompatibility complex antigen in NIH 3T3 cells. In addition, these vectors were used to express DR antigens in two human mutant B-lymphoblastoid cell lines, one of which was deleted for both alleles of the DR alpha gene and the other of which expressed no class II antigens because of a genetic defect in a putative trans-acting regulatory factor.
Mol Cell Biol 1987 Nov
PMID:Expression of HLA-DR antigen in human class II mutant B-cell lines by double infection with retrovirus vectors. 282 20

An amphotropic retroviral vector containing the bacterial neomycin phosphotransferase gene (neo) was used to infect blast cells from patients with acute myeloblastic leukemia. The infected cells acquired a G418-resistant phenotype that was stable as measured in a clonogenic assay and in long-term suspension culture. Thus, gene transfer into stem cells was accomplished by this procedure. This approach for manipulating gene expression in blast stem cells provides a means to assess the roles of a variety of genes in self-renewal, differentiation, and leukemogenesis.
Mol Cell Biol 1988 Feb
PMID:Introduction of new genetic material into human myeloid leukemic blast stem cells by retroviral infection. 283 45

The human cell line LM205, transformed with the pLR309 plasmid, contains a stably integrated selectable gene marker (neo) without a transcriptional promoter. Spontaneous tandem duplication at the integration site relocates a Simian virus 40 transcriptional promoter to a position 5' to the neo gene at a rate of 5 x 10(-8) events/cell/generation, as measured by subsequent resistance of the cells to the toxic antibiotic G418. The heterogeneity in the site of recombination observed in various G418-resistant (G418-R) subclones indicates that the sequences involved have little or no homology. The rate of tandem duplication involving the neo gene was not affected by DNA-damaging agents or by inhibitors of DNA synthesis. Although these tandem duplications were relatively stable in most G418-R subclones, others underwent further amplification of the neo gene during cloning. In one such cell line, RS-4, subclones isolated without G418 demonstrated a high degree of heterogeneity in the neo gene copy number (2-20), indicating that amplification was associated with a high rate of homologous recombination. Because LM205 was the only clone out of the 30 original clones transformed with pLR309 that demonstrated spontaneous G418-R colonies, cell DNA sequences near the integrated neo gene may promote this recombination. Inclusion of this cell DNA in the initial tandem duplication might then explain the high rate of duplication and deletion observed in the region of the neo gene in the RS-4 subclone.
Somat Cell Mol Genet 1988 May
PMID:Association of high rate of recombination with amplification of dominant selectable gene in human cells. 283 23

Expression of the biologically active beta-subunit of mouse nerve growth factor (beta-NGF) was conferred onto cultured AtT-20 mouse pituitary cells via a replication-defective retroviral vector. The retroviral LTR promoter was used for expression of a cDNA for beta-NGF corresponding to the shorter mRNA species produced by most tissues that receive sympathetic innervation. The vector included the TU5 gene conferring resistance to the neomycin analogue G418 under the control of an SV40 early promoter. AtT-20 cells, which produce essentially no endogenous beta-NGF, were infected and then cloned under G418 selection. Clones were evaluated for release into the medium of biologically active beta-NGF using a bioassay for neurite extension from PC-12 cells. The biological activity was equivalent to 1 to 10 ng of beta-NGF per mg cell protein over 24 hours. Immune precipitation and SDS/polyacrylamide gel electrophoresis of labelled proteins in the medium showed that the major form of immunoreactive beta-NGF secreted from cells comigrated with authentic mature beta-NGF, apparent Mr 13,000. Release of this beta-NGF from cells was stimulated by addition of 1 mM-8-bromocyclic AMP or 10 nM-corticotropin releasing factor, suggesting that at least some of the processed factor is stored in secretory vesicles. These studies, together with those on other cultured cells, which produce beta-NGF and lack secretory granules, e.g. L cells, suggest that the beta-NGF precursor synthesized from the shorter mRNA species can be processed and secreted through either the regulated or constitutive route. This retroviral vector provides a potential means of conferring beta-NGF expression onto a number of different cell types in culture and in vivo.
Mol Biol Med 1988 Feb
PMID:Retrovirus-mediated gene transfer of beta-nerve growth factor into mouse pituitary line AtT-20. 283 91

Phycomyces protoplasts transformed with a plasmid containing the bacterial gene for kanamycin resistance grow in the presence of G418, a kanamycin analogue. The plasmid also contains a Phycomyces DNA sequence that supports autonomous replication in yeast. We obtained about 250 transformants per microgram DNA or one per 5000 viable protoplasts. The transformant phenotype is retained under selective conditions and lost in the majority of the vegetative spores. Recovered plasmids and Southern analysis indicate that the plasmid probably replicates autonomously in Phycomyces.
Mol Gen Genet 1988 Apr
PMID:Transformation of Phycomyces with a bacterial gene for kanamycin resistance. 283 99

Retroviral vectors were constructed which coexpressed three inserted genes from independent transcriptional promoters in singly infected cells. Several such triple-promoter vectors were constructed with various combinations of oncogenes and selectable drug resistance genes. All expressed three mRNAs of the expected size in infected cells. One vector expressing the v-Ha-ras, v-myc, and neo genes was characterized in detail. This retrovirus did not undergo rearrangement during the process of infection, as judged by Southern analysis, and infection of primary rat embryo fibroblasts demonstrated that ras-myc-cotransformed cells could be selected in G418. This demonstration that retroviral vectors can be used to express three cistrons independently increases their value as gene transfer vehicles, particularly for studies involving oncogene cooperation in primary cells.
Mol Cell Biol 1988 Apr
PMID:Stably transmitted triple-promoter retroviral vectors and their use in transformation of primary mammalian cells. 283 55

Two plasmids encoding resistance to hygromycin-B or to the analog of neomycin, G418, and containing either one or two copies of the plasmid origin of replication, oriP, of Epstein-Barr virus (EBV) were introduced into an EBV-positive B-lymphoblastoid cell line. Two clones of cells containing both plasmids were analyzed for the number of copies of each plasmid when the cells were propagated in the absence or in the presence of one or both selective agents. Under all conditions tested, the plasmid with two copies of oriP behaved in cells as did the plasmid with one copy of this plasmid origin of replication.
Mol Biol Med 1988 Apr
PMID:Plasmid origin of replication of Epstein-Barr virus, oriP, does not limit replication in cis. 284 May 51

We examined the ability of unlinked nonreplicating plasmid molecules to undergo homologous recombination during cotransformation of Dictyostelium amoebae. The transformation vector B10S confers resistance to the antibiotic G418 and was always presented to amoebae as a closed circle. Cotransforming DNA, containing a slime mold cDNA and sequences homologous to the primary vector, was presented either as a closed circle or as a linear molecule after digestion with restriction endonucleases which cut within one of three distinct regions of the plasmid. Remarkably, homologous recombination occurred in every clone examined. Moreover, the products of recombination were identical in all instances, irrespective of the presence or position of linearized ends. The ends of the linear templates were not recombinogenic. Repair of the introduced double-strand break occurred frequently during recombination. The repair could occur intermolecularly or, more likely, intramolecularly, i.e., by recircularization. Many of the recombination events were of a nonreciprocal nature. Despite the startlingly frequent level of homologous recombination, the use of cotransforming DNA which contains no homology to the selected vector established that such recombination was not required for cotransformation.
Mol Cell Biol 1988 Jul
PMID:Homologous recombination and the repair of double-strand breaks during cotransformation of Dictyostelium discoideum. 284 87

Activated ras oncogenes have previously been implicated in the pathogenesis of human lung carcinomas. A v-Ha-ras-containing retrovirus, Zip-ras, was generated by inserting the coding region of the v-Ha-ras oncogene into the Zip-NeoSV(X) [Cepko et al., Cell 37:1053-1062, 1984] retroviral vector. Amphotrophic Zip-ras retrovirus was used to infect an SV40 large T antigen-positive immortalized cell line, BEAS-2B, derived from normal bronchial epithelial cells, the predominant progenitor cells of human lung carcinomas. Zip-ras-infected BEAS-2B cells selected for G418 resistance formed anaplastic carcinomas in 12 of 15 athymic nude mice (latency 3 wk), whereas Zip-NeoSV(X)-infected BEAS-2B control cultures inoculated into 12 nude mice formed no tumors after a minimum of 7 mo. Tumor cell lines were established and demonstrated to be of human epithelial origin and to express v-Ha-ras p21 protein. A common feature of the tumor cell lines was an increase in ploidy. The increased efficiency of neoplastic transformation by v-Ha-ras of cell lines as compared with our previous results with normal bronchial epithelial cells [Yoakum et al., Science 227:1174-1179, 1985] is consistent with the hypothesis that the "immortalization" step is rate-limiting in in vitro human epithelial cell carcinogenesis.
Mol Carcinog 1988
PMID:Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding viral Harvey ras. 285 21

Two amphotropic-based mouse retroviral vectors carrying the neomycin-resistance gene were used to infect four bovine cell lines. Two cell lines, bovine kidney and spleen cells, were refractory to the infection while two independent bovine cells of apparent embryonic origin were infected by the amphotropic retroviral vectors at a measurable titer. Southern blot analysis reveals the presence of neomycin-resistance gene in the G418-resistant bovine cells. The results demonstrate the successful transfer of a gene to bovine cells of embryonic origin using a murine retroviral vector system.
Somat Cell Mol Genet 1989 Mar
PMID:Infection of bovine cells of embryonic origin by amphotropic retroviral vectors. 292 39


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