Chromosome-mediated gene transfer results in two classes of unstable transformants.

Klobutcher, Lawrence A.; Miller, Carol; Ruddle, Frank H.

doi:10.1073/pnas.77.6.3610

Cited by 51 publications

(25 citation statements)

References 28 publications

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“…Here, we studied the introduction of the 21DpqHAC vector into normal hPFs with MMCT, 7,8 which is recognized as the most suitable method for the transfer of a single, intact chromosome from one cell to another and enabled the lower incidence of fragmentation and rearrangement of donor chromosomes compared to transfection-based methods. 9,10 To our knowledge, this is the first report to demonstrate that the structurally defined, extra artificial chromosome is highly maintained in normal hPFs without accompanying aberration of host chromosomes. In the resultant hPFs carrying the HAC vector, we also showed the long-term expression of therapeutic transgene, human erythropoietin (EPO) as a model, which is a growth factor for erythroid cells and widely used for the clinical treatment of anemia in renal insufficiency.…”

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confidence: 99%

Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts

et al. 2005

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confidence: 99%

Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts

et al. 2005

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“…Phenotypic stabilization due to extended selection has been reported after DNA-or chromosome-mediated gene transfer (Degnen et al, 1976;Willecke et al, 1976Willecke et al, , 1979Klobutcher et al, 1981;Scangos et al, 1981) and has been shown to be due to integration of the transgenomes into recipient chromosomes (Perucho et al, 1980;Robins et al, 1981). Recently, Murray et al (1983) showed that a transferred transforming gene was segregated from transformants when the selection pressure was lifted.…”

Section: Resultsmentioning

confidence: 99%

Unstable transformation of mouse 3T3 cells by transfection with DNA from normal human lymphocytes.

Schäfer¹,

Griegel²,

Dubbert³

et al. 1984

The EMBO Journal

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The corresponding primary transformants were not tumorigenic, however, and the ability to proliferate in semi-solid agar medium was gradually lost when the cells were grown as non-confluent monolayers. Furthermore, in contrast to secondary transformants, DNA from primary transformants showed only relatively weak hybridization to a human Alu repetitive DNA probe. We conclude that in primary transformants the transformed phenotype is expressed in an unstable fashion whereas secondary transformants appear to be stably transformed.

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“…Table 2 Since instability of various transfected phenotypes (13,20,28) has been reported, we investigated the stability of the P+ phenotype after transfection. After transfection of P+ DNA into JB6 Cl 30 cells, these cells were induced by TPA to form soft agar colonies.…”

Section: Resultsmentioning

confidence: 99%

Transfer of Sensitivity to Tumor Promoters by Transfection of DNA from Sensitive into Insensitive Mouse JB6 Epidermal Cells

Colburn¹,

Talmadge²,

Gindhart³

1983

Mol. Cell. Biol.

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Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P+) and nonpromotable (P-) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12-o-tetradecanoyl-phorbol-13-acetate (TPA). Cellular DNA isolated from one of two P+ lines, JB6 Cl 41 or JB6 Cl 22, was CaPO4 precipitated and used to transfect the P-cell line JB6 Cl 30. At 7 days posttransfection, the cells were suspended in agar with or without TPA at 1.6 x 10-8 M and assayed 10 days later for TPA-dependent colony formation. Whether there exists a genetic basis for susceptibility to tumor promoters is one of the important unanswered questions in carcinogenesis. Answering this question with in vivo initiation-promotion models (1, 3) has been difficult because tumor promotion is assumed to occur in a minor subpopulation consisting of carcinogeninitiated or post-initiated cells. Because such "promotable" subpopulations of carcinogen-exposed cells have not yet been identified in animals, one must study heterogeneous populations. For studying the genetics of tumor promoter sensitivity, clonal lines of initiated or post-initiated cells in culture can be useful. To be analogous to the mouse skin initiation-promotion model, initiated cells should have two essential features: (i) they must be stably nonneoplastic, and (ii) they must require exposure only to a tumor promoter, not to a complete carcinogen, to become irreversibly neoplastic. Postinitiated cells, i.e., those which have undergone early-stage promotion (10, 24) should differ from initiated cells by requiring a relatively short period of exposure to tumor promoter. The mouse JB6 epidermal cell model which we have developed satisfies the criteria indicated above for post-initiated cells.JB6 cells, originally established as a nonclonal line from untreated primary BALB/c mouse epidermal cultures, are nontumorigenic and anchorage dependent (7) and retain an epidermal specific cell surface antigen (2). Exposure of these cells to phorbot esters and other tumor promoters irreversibly promotes anchorage independence and tumorigenicity (4). Promotion of anchorage independence occurs when JB6 cells are exposed to second-stage (24, 25) but not first-stage tumor promoters and is inhibited by second-stage, not first-stage, promotion inhibitors (8). The JB6 model system thus appears to 1182 on May 9, 2018 by guest

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Chromosome-mediated gene transfer results in two classes of unstable transformants.

Abstract: The human thymidine kinase gene has been transferred from HeLa S3 cells to mouse LM (TK-)

Cited by 51 publications

References 28 publications

Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts

Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts

Unstable transformation of mouse 3T3 cells by transfection with DNA from normal human lymphocytes.

Transfer of Sensitivity to Tumor Promoters by Transfection of DNA from Sensitive into Insensitive Mouse JB6 Epidermal Cells

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