文章基本信息

标题：Transfer of the human gene for hypoxanthine-guanine phosphoribosyltransferase via isolated human metaphase chromosomes into mouse L-cells
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作者：K Willecke ; F H Ruddle
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1975
卷号：72
期号：5
页码：1792-1796
DOI：10.1073/pnas.72.5.1792
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We have transferred the human gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8 ; IMP:pyrophosphate phosphoribosyltransferease) via isolated metaphase chromosomes from human HeLa S3 cells into murine A9 cells which lack functional murine HPRT activity, using the technique of McBride and Ozer (Proc, Nat. Acad. Sci. USA 70, 1258-1262, 1973). Three transformed clones were isolated which contained human HPRT activity as determined by electrophoretic and immunochemical assays. Twenty human isozymes other than HPRT whose genes have been assigned to 14 human chromosomes were found to be absent in our transformed clones. Moreover, the human isozymes of hlucose-6-phosphate dehydrogenase (EC 1.1.1.49 ; D-glucose 6-phosphate:NADP 1-oxidoreductase) and phosphoglycerate kinase (EC 2.7.2.3 ;ATP:3-phospho-D-glycerate 1-phosphotransferase), whose genes have been linked with the HPRT gene to the long are of the human X chromosome, were also absent. On the basis of the known linkage relationships of the three markers, we thereby suggest that the transferred piece of human genetic material is smaller than 20% of the human X chromosome or less than 1% of the human genome. This estimate assumes a normal syntenic relationship for the long arm of the X chromosome in HeLa S3 cells. In agreement with this conclusion, no human chromosomes could be detected in our transformed clones. When grown under nonselective conditions about 3% of the gene transfer cells lost the human HPRT marker per cell generation. Transformants that had lost human HPRT activity were subjected to hypoxanthine-aminopterin-thymidine selection. The frequency of revertants to the HPRT(+) phenotype was less than 1 x 10(-6), and two revertants that were obtained possessed the mouse electrophoretic phenotype. These results argue against a stable integration of the human donor genetic material into the mouse recipient genome.