Molecular cloning of MER-2, a human chromosome-11-encoded red blood cell antigen, using linkage of cotransfected markers
- 1 September 1987
- journal article
- research article
- Published by Springer Nature in Somatic Cell and Molecular Genetics
- Vol. 13 (5) , 553-561
- https://doi.org/10.1007/bf01534496
Abstract
We report the molecular cloning of a human gene MER-2located on chromosome 11 that encodes a cell surface antigen which is polymorphic on red blood cells. An essential element of the cloning strategy was cotransfection-induced linkage of pSV2-neo, which encodes resistance to the antibiotic G418, to the human MER-2gene. An important feature of the pSV2-neo construct is that the same gene (the transposon, Tn5) that encodes G418 resistance in eukaryotic cells confers neomycin resistance in bacteria. Chinese hamster ovary (CHO) cells were cotransfected with pSV2-neo and genomic DNA from a CHO ×human cell hybrid containing a single human chromosome (chromosome 11). Transfectants expressing both the human MER-2gene and G418 resistance were isolated by selection in the antibiotic G418, followed by indirect immunofluorescence using the monoclonal antibody 1D12, which recognizes the MER-2 antigen, manual enrichment, and single-cell cloning. Genomic DNA from a primary transfectant positive for MER-2expression and G418 resistance was used to construct a cosmid library and cosmid clones able to grow in neomycin were isolated. Of 150,000 cosmid clones screened, 90 were resistant to neomycin and of these, 11 contained human repetitive sequences. Five neomycin-resistant cosmid clones containing human repetitive DNA were able to transfect CHO cells for G418 resistance and MER-2expression.This publication has 20 references indexed in Scilit:
- MER2: A Red Cell Polymorphism Defined by Monoclonal Antibodies1Vox Sanguinis, 1987
- The isolation and sequence of the gene encoding T8: A molecule defining functional classes of T lymphocytesCell, 1985
- Report of the committee on the genetic constitution of chromosomes 10, 11, and 12Cytogenetic and Genome Research, 1985
- Gene transfer, expression, and molecular cloning of the human transferrin receptor geneCell, 1984
- Expression of human myeloid-associated surface antigens in human-mouse myeloid cell hybrids.Proceedings of the National Academy of Sciences, 1983
- Stable transformation of mouse L cells for human membrane T-cell differentiation antigens, HLA and beta 2-microglobulin: selection by fluorescence-activated cell sorting.Proceedings of the National Academy of Sciences, 1983
- Genetic and biochemical analysis of the a1 cell-surface antigen associated with human chromosome 11.Proceedings of the National Academy of Sciences, 1979
- DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells.Proceedings of the National Academy of Sciences, 1979
- Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donorCell, 1978
- Transfer of purified herpes virus thymidine kinase gene to cultured mouse cellsCell, 1977