Selection of Hybrids from Matings of Fibroblasts in vitro and Their Presumed Recombinants

Littlefield, John W.

doi:10.1126/science.145.3633.709

Cited by 2,050 publications

(543 citation statements)

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“…Cells were exposed to the DNA precipitate for 24 h, at which time they were fed fresh MEM-10% fetal calf serum. After an additional 24 h, the cells were challenged with hypoxanthine-aminopterinthymidine (HAT) medium to select for TK+ transformants (14). Transfections to test for the ability of A27 and B2-1 cells to be transformed were carried out as previously described (11).…”

Section: Methodsmentioning

confidence: 99%

Isolation of a simian virus 40 T-antigen-positive, transformation-resistant cell line by indirect selection.

Ryan¹,

Christensen²,

Imperiale³

et al. 1985

Mol. Cell. Biol.

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In an attempt to identify cellular genes that might be involved in simian virus 40 (SV40) transformation, we have set out to isolate cells which express T antigen but are not transformed. SV40 DNA and the herpes simplex virus thymidine kinase gene were cotransfected into tk-3T3 fibroblasts. Of 72 colonies screened that were resistant to hypoxanthine-aminopterin-thymidine, 57 were T antigen positive as judged by immunofluorescence. One of these lines, A27, had a normal growth phenotype in monolayer overgrowth and soft agar assays. It contained intact SV40 sequences that could be rescued by fusion to permissive cells. This rescued virus was fully capable of transforming nonpermissive cells to the same extent as did wild-type virus. The A27 cells, however, were not transformable by infection with SV40 or by transfection of SV40 DNA. It is likely that these cells were altered in a cellular function required for the establishment of the transformed state.

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Section: Methodsmentioning

confidence: 99%

Isolation of a simian virus 40 T-antigen-positive, transformation-resistant cell line by indirect selection.

Ryan¹,

Christensen²,

Imperiale³

et al. 1985

Mol. Cell. Biol.

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“…The cell suspension was washed and resuspended in 40 ml of complete RPMI 1640 (20% foetal calf serum, Seromed, Miunchen, Germany) and distributed on 4 96-well, flat-bottomed microtitre plates (Falcon). On Days 1, 2, 4, 7 and 11 the culture medium was replaced with conditioned hypoxanthine-aminopterin-thymidine (HAT) medium (Littlefield, 1964). Two to three weeks after fusion, 100 ,l of culture supernatant from wells with growing hybrids, as seen under the microscope, were collected for preliminary screening.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a monoclonal antibody to L1210 leukaemia

Testorelli¹,

Morelli²,

Goldin³

et al. 1982

Br J Cancer

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Summary.-A mouse monoclonal cell line (L1) was produced by fusing the mouse myeloma P3X63/Ag8 with CD2F1 spleen cells immunized with a highly immunogenic subline of L1210 leukaemia (Li210/DTIC). A very few positive clones (1%) were isolated and one of these was chosen for detailed study. The monoclonal antibody Li is an IgM immunoglobulin strongly reacting in a complement-dependent cytotoxicity assay against L1210/Cr leukaemia and its more or less inmmunogenic sublines. The specificity of the LI antibody against L1210 leukaemia was studied by extensive screening with normal adult and foetal tissues, lymphoid tissues from several independent strains and a panel of the most common experimental tumours, to all of which it was unreactive. Attempts at immunotherapy were carried out in DBA/2 mice challenged with L1210 leukaemia and treated with Li (ascites) and complement. Although the in vitro cytotoxic titre of ascites fluid from mice bearing hybridoma was very high (10-7), no therapeutic effect was obtained in vivo.

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“…For the fusion we used polyethyleneglycol (PEG-l 000). The PEG-treated cells were seeded into 24-well Costar plates, and hybrids were selected in growth medium containing 100 PM hypoxanthine, 0.4 PM aminopterin, and 16 I.IM thymidine (HAT medium) [19]. Hybrids producing mouse immunoglobulins were screened by radioimmunoassay (RIA) [ 16,171 and grown in the culture medium for the classification of mouse immunoglobulins or were injected intraperitoneally into mice for further growth and production of antibody in the ascites fluid.…”

Section: Methodsmentioning

confidence: 99%