An approach to the cloning of cell surface protein genes. Selection by cell sorting of mouse L-cells that express HLA or 4F2 antigens after transformation with total human DNA - PubMed (original) (raw)

• PMID: 6680152

An approach to the cloning of cell surface protein genes. Selection by cell sorting of mouse L-cells that express HLA or 4F2 antigens after transformation with total human DNA

L C Kühn et al. Mol Biol Med. 1983 Oct.

Abstract

We describe an approach to the cloning of cell surface proteins that is independent of messenger RNA isolation. Mouse Ltk- cells are cotransformed with the thymidine kinase gene from Herpes Simplex Virus and total human DNA. Transformants expressing the human surface antigens of interest are isolated by two selection steps, consisting of treatment with hypoxanthine/aminopterin/thymidine and fluorescence-activated cell sorting. Using this procedure, we isolated seven transformants expressing HLA-A,B,C antigens and 12 transformants expressing the 4F2 antigen. We have so far failed to identify any OKT-10 antigen expressing L-cell transformants. Three independent secondary 4F2 transformants were obtained after identical cotransformation of fresh Ltk- cells with DNA from primary transformants. Analysis of their genome by hybridization with human DNA revealed a shared set of human restriction fragments in all three cell lines. This 32 X 10(3) base-pair segment of DNA codes for the human 4F2 antigen, thereby offering the opportunity to clone the gene. To substantiate this hypothesis, we analyzed the seven HLA-expressing cell lines, and we found that all of them had acquired an HLA-coding sequence concomitant to its expression.

PubMed Disclaimer

Similar articles

• Somatic cell genetics and flow cytometry.
  Kamarck ME, Barbosa JA, Kühn L, Peters PG, Shulman L, Ruddle FH. Kamarck ME, et al. Cytometry. 1983 Sep;4(2):99-108. doi: 10.1002/cyto.990040202. Cytometry. 1983. PMID: 6354642 Review.
• Stable transformation of mouse L cells for human membrane T-cell differentiation antigens, HLA and beta 2-microglobulin: selection by fluorescence-activated cell sorting.
  Kavathas P, Herzenberg LA. Kavathas P, et al. Proc Natl Acad Sci U S A. 1983 Jan;80(2):524-8. doi: 10.1073/pnas.80.2.524. Proc Natl Acad Sci U S A. 1983. PMID: 6188154 Free PMC article.
• Identification of human genomic clones coding the major histocompatibility antigens HLA-a2 and HLA-B7 by DNA-mediated gene transfer.
  Barbosa JA, Kamarck ME, Biro PA, Weissman SM, Ruddle FH. Barbosa JA, et al. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6327-31. doi: 10.1073/pnas.79.20.6327. Proc Natl Acad Sci U S A. 1982. PMID: 6959120 Free PMC article.
• Analysis of the expression of cloned HLA class I genes in mouse transfected L cells by quantitative flow cytometry.
  Le Bouteiller PP, N'Guyen C, Lemonnier FA, Jordan BR, Caillol D, Mishal Z. Le Bouteiller PP, et al. Cytometry. 1985 Sep;6(5):414-21. doi: 10.1002/cyto.990060505. Cytometry. 1985. PMID: 4042783

Cited by

• Functional expression of a human Na+/H+ antiporter gene transfected into antiporter-deficient mouse L cells.
  Franchi A, Perucca-Lostanlen D, Pouyssegur J. Franchi A, et al. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9388-92. doi: 10.1073/pnas.83.24.9388. Proc Natl Acad Sci U S A. 1986. PMID: 3025840 Free PMC article.
• Gene transfection of the HuLy-m2 (Leu-9) antigen into mouse L cells.
  Sandrin MS, Lovering KE, Tachas G, Collins PR, McKenzie IF. Sandrin MS, et al. Immunogenetics. 1987;25(5):279-83. doi: 10.1007/BF00404419. Immunogenetics. 1987. PMID: 3106199
• The William Allan Memorial Award address: Reverse genetics and beyond.
  Ruddle FH. Ruddle FH. Am J Hum Genet. 1984 Sep;36(5):944-53. Am J Hum Genet. 1984. PMID: 6594045 Free PMC article. No abstract available.
• The internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information.
  Dargemont C, Le Bivic A, Rothenberger S, Iacopetta B, Kühn LC. Dargemont C, et al. EMBO J. 1993 Apr;12(4):1713-21. doi: 10.1002/j.1460-2075.1993.tb05816.x. EMBO J. 1993. PMID: 8467813 Free PMC article.
• Noncoding 3' sequences of the transferrin receptor gene are required for mRNA regulation by iron.
  Owen D, Kühn LC. Owen D, et al. EMBO J. 1987 May;6(5):1287-93. doi: 10.1002/j.1460-2075.1987.tb02366.x. EMBO J. 1987. PMID: 3608980 Free PMC article.

Publication types

MeSH terms

Substances

LinkOut - more resources

• Other Literature Sources

  • The Lens - Patent Citations Database

• Research Materials

  • NCI CPTC Antibody Characterization Program