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1.
Two dominant-acting selectable markers for gene transfer studies in mammalian cells. 总被引:37,自引:5,他引:37
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S C Hartman R C Mulligan 《Proceedings of the National Academy of Sciences of the United States of America》1988,85(21):8047-8051
We report the development of two dominant-acting genetic markers useful for monitoring gene transfer in mammalian cells that are based on prokaryotic genes encoding key steps in the synthesis of the essential amino acids, tryptophan and histidine. Under appropriate conditions, expression of these genes obviates the nutritional requirements for their respective amino acid products. Expression of the trpB gene of Escherichia coli, which encodes the beta subunit of tryptophan synthase (EC 4.2.1.20), allows mammalian cell survival and multiplication in medium containing indole in place of tryptophan. The hisD gene of Salmonella typhimurium encodes histidinol dehydrogenase (EC 1.1.1.23), which catalyzes the two-step NAD+-dependent oxidation of L-histidinol to L-histidine. In medium lacking histidine and containing histidinol, only mammalian cells expressing the hisD product survive. The selection is a double one in that the provided precursor histidinol is itself toxic to animal cells through its inhibition of histidyl-tRNA synthetase; thus, the dehydrogenase both removes an inhibitor and forms a required end product. Alternatively, the his selection may be carried out under conditions in which the dehydrogenase serves mainly to detoxify histidinol. For either the trp or his selections the substitute nutrient (indole or histidinol) is readily available, inexpensive, stable, permeable to cells, and convertible to the end product in a step controlled by a single gene. Vectors based upon murine retrovirus and papovavirus backbones have been successfully employed for both genes, allowing selection in a range of cell types, including 3T3, CV-1, and HeLa. These dominant selective schemes should provide generally useful and inexpensive alternatives to others currently in use, such as the gpt, neo, hygro, dhfr, and tk selections. 相似文献
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C J Bieberich M F Utset A Awgulewitsch F H Ruddle 《Proceedings of the National Academy of Sciences of the United States of America》1990,87(21):8462-8466
The region-specific patterns of expression of mouse homeobox genes are considered important for establishing the embryonic body plan. A 5-kilobase (kb) DNA fragment from the Hox-3.1 locus that is sufficient to confer region-specific expression to a beta-galactosidase reporter gene in transgenic mouse embryos has been defined. The observed reporter gene expression pattern closely parallels endogenous Hox-3.1 expression in 8- to 9.5-day postcoitum (p.c.) embryos. At 10.5 days p.c. and later, the pattern of beta-galactosidase activity diverges from the Hox-3.1 pattern, and an inappropriately high level of reporter gene expression is observed in posterior spinal ganglia. Inclusion of an additional 2 kb of upstream sequences is sufficient to suppress this aberrant expression in the developing spinal ganglia. Together, these results show that the control of early Hox-3.1 expression is complex, involving at least one positively acting and one negatively acting element. 相似文献
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F M Boyce N L Bucher 《Proceedings of the National Academy of Sciences of the United States of America》1996,93(6):2348-2352
This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells. 相似文献
4.
Ecdysone-inducible gene expression in mammalian cells and transgenic mice. 总被引:23,自引:0,他引:23
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D No T P Yao R M Evans 《Proceedings of the National Academy of Sciences of the United States of America》1996,93(8):3346-3351
During metamorphosis of Drosophila melanogaster, a cascade of morphological changes is triggered by the steroid hormone 20-OH ecdysone via the ecdysone receptor, a member of the nuclear receptor superfamily. In this report, we have transferred insect hormone responsiveness to mammalian cells by the stable expression of a modified ecdysone receptor that regulates an optimized ecdysone responsive promoter. Inductions reaching 4 orders of magnitude have been achieved upon treatment with hormone. Transgenic mice expressing the modified ecdysone receptor can activate an integrated ecdysone responsive promoter upon administration of hormone. A comparison of tetracycline-based and ecdysone-based inducible systems reveals the ecdysone regulatory system exhibits lower basal activity and higher inducibility. Since ecdysone administration has no apparent effect on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene in transgenic mice and for gene therapy. 相似文献
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Homozygosity for a dominant negative thyroid hormone receptor gene responsible for generalized resistance to thyroid hormone. 总被引:1,自引:0,他引:1
S Ono I D Schwartz O T Mueller A W Root S J Usala B B Bercu 《The Journal of clinical endocrinology and metabolism》1991,73(5):990-994
Generalized resistance to thyroid hormones (GRTH) commonly results from mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene. We have reported on a novel deletion mutation in c-erbA beta in a kindred, S, with GRTH. One patient from this kindred was the product of a consanguineous union from two affected members and was homozygous for the beta-receptor defect. This patient at 3.5 weeks of age had unprecedented elevations of TSH, free T4, and free T3 (TSH, 389 mU/L; free T4, 330.8 pmol/L; free T3, 82,719 fmol/L). He displayed a complex mixture of tissue-specific hyperthyroidism and hypothyroidism. He had delayed growth (height age, 1 3/12 yr at chronological age 2 9/12 yr) and skeletal maturation (bone age, 4 months), and developmental delay (developmental age, 8 months), but he was quite tachycardic. The homozygous patient of kindred S is markedly different from a recently reported patient with no c-erbA beta-receptor. This difference indicates that a dominant negative form of c-erbA beta in man can inhibit at least some thyroid hormone action mediated by the c-erbA alpha-receptors. 相似文献
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Cell-specified expression of a selectable hybrid gene. 总被引:21,自引:2,他引:21
V Episkopou A J Murphy A Efstratiadis 《Proceedings of the National Academy of Sciences of the United States of America》1984,81(15):4657-4661
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Positive genetic selection for gene disruption in mammalian cells by homologous recombination. 总被引:18,自引:3,他引:15
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J M Sedivy P A Sharp 《Proceedings of the National Academy of Sciences of the United States of America》1989,86(1):227-231
Efficient modification of genes in mammalian cells by homologous recombination has not been possible because of the high frequency of nonhomologous recombination. An efficient method for targeted gene disruption has been developed. Cells with substitution of exogenous sequences into a chromosomal locus were enriched, by a factor of 100, using a positive genetic selection that specifically selects for homologous recombination at the targeted site. The selection is based on the conditional expression of a dominant selectable marker by virtue of in-frame gene fusion with the target gene. The dominant selectable marker was derived by modification of the Escherichia coli neo gene so that it retains significant activity in mammalian cells after in-frame fusion with heterologous coding sequences. In the example presented here, homologous recombinants were efficiently recovered from a pool in which the targeted gene was disrupted in 1 per 10,000 cells incorporating exogenous DNA. 相似文献
11.
Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. 总被引:29,自引:6,他引:29
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D A Nielsen J Chou A J MacKrell M J Casadaban D F Steiner 《Proceedings of the National Academy of Sciences of the United States of America》1983,80(17):5198-5202
As an approach to the study of mammalian gene expression, the promoters and translation initiation regions of the rat preproinsulin II and the simian virus 40 early genes were fused to the structural gene of Escherichia coli beta-galactosidase, a sensitive probe for gene expression. These fusions were introduced into COS-7 cells, a simian virus 40 large tumor-antigen-producing monkey kidney cell line, where they directed the synthesis of enzymatically active hybrid beta-galactosidase proteins. Conditions for transfection were varied to optimize the expression of beta-galactosidase activity in the transfected cells. The pH optimum of this activity was found to be 7.0, the same as that of native E. coli beta-galactosidase and distinct from the major lysosomal "acid" beta-galactosidase. The fused preproinsulin-beta-galactosidase was further characterized by gel electrophoresis of nondenatured cell extracts stained by a fluorogenic substrate and by immunoprecipitation and gel electrophoresis of 3H-labeled cell proteins. These results all indicate that fully active tetrameric beta-galactosidase hybrids can be produced in mammalian cells. The expression of preproinsulin-beta-galactosidase activity was measured in the presence of high glucose, insulin, dexamethasone, or epidermal growth factor but no regulatory changes were observed. 相似文献
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A L Greenleaf 《Proceedings of the National Academy of Sciences of the United States of America》1993,90(23):10896-10897
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P Upcroft H Skolnik J A Upcroft D Solomon G Khoury D H Hamer G C Fareed 《Proceedings of the National Academy of Sciences of the United States of America》1978,75(5):2117-2121
The transduction of an Escherichia coli gene into mammalian cells is described. A supressor tRNA gene was linked to a simian virus 40 (SV40) vector in vitro and the recombinant was used to transfect rat embryo cells and monkey kidney cells. The hybrid SV40 genome, SV40-su+ III, retained genetic information required for autonomous replication and cellular transformation and had a 1300-base-pair DNA segment in the late gene region (between the restriction endonuclease sits Hpa II at 0.735 and EcoRI at 0/1.0 on the SV40 genetic map) replaced by an 870-base-pair bacterial DNA segment containing the suppressor tRNA gene, su+ III (tRNATyrsu+III). The structure and fate of the SV40-su+III chimera were determined by DNA reassociation kinetic analysis and restriction enzyme cleavage of the total cellular DNA from transformed rat embryo cells and persistently infected monkey cells. Hybridization with radiolabeled probes specific for vector (SV40) or su+III DNA sequences revealed primarily nonintegrated or free hybrid genomes. In cloned lines of both cell types, the bacterial DNA segment was recovered intact, as judged by the length of the segment excised by restriction endonucleases and its ability to hybridize to the radiolabeled bacterial DNA probe and not to the SV40 probe. 相似文献
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Induction of apoptosis in K562 cells by dominant negative c-myb 总被引:2,自引:0,他引:2
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Kim DH Saetrom P Snøve O Rossi JJ 《Proceedings of the National Academy of Sciences of the United States of America》2008,105(42):16230-16235
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Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system. 总被引:17,自引:3,他引:17
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C A Mullen M Kilstrup R M Blaese 《Proceedings of the National Academy of Sciences of the United States of America》1992,89(1):33-37
Expression of the bacterial gene for cytosine deaminase (CD; EC 3.5.4.1) in mammalian cells was evaluated as a negative selection system or suicide vector for potential use in gene transfer studies and therapies. Mammalian cells, unlike certain bacteria and fungi, do not contain the enzyme CD and do not ordinarily metabolize cytosine to uracil. Nor do they metabolize the innocuous compound 5-fluorocytosine to the highly toxic compound 5-fluorouracil. The Escherichia coli CD gene underwent PCR oligonucleotide-directed mutagenesis to enhance its expression in a eukaryotic system and it was then cloned into an expression vector, pLXSN, that also contains a neomycin-resistance gene. Murine fibroblast lines were transfected with the plasmid and subjected to brief selection in the neomycin analogue G418. Lysates from these cell populations exhibited significant CD activity detected by conversion of radiolabeled cytosine to uracil. In clonogenic assays transfected cells expressing CD were selectively killed by incubation in 5-fluorocytosine, whereas control cell lines were not. Dose-response studies evaluating [3H]thymidine incorporation or cloning efficiency demonstrated profound inhibition at and above 65 micrograms of 5-fluorocytosine per ml. Mixed cellular assays showed that CD-positive cells could be eliminated without bystander killing of other cells. Retrovirus-mediated CD gene transfer into various tissues was also demonstrated. Thus CD, with its ability to produce the toxic antimetabolite 5-fluorouracil from 5-fluorocytosine, may be useful as a negative selection system for studies and treatments employing gene transfer techniques. 相似文献