Structure of thymidine kinase gene introduced into mouse Ltk- cells by a new injection method |
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| Authors: | A Kudo F Yamamoto M Furusawa A Kuroiwa S Natori M Obinata |
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| Institution: | 1. Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, TokyoJapan;1. Department of Biology, Faculty of Science, Osaka City University, OsakaJapan |
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| Abstract: | Pricking, a new injection method developed by Yamamoto et al. (1981), can be used to introduce DNA into cultured cells with high efficiency. Closed circular plasmid DNA containing the cloned HSV-TK gene (pTK-1) was introduced by this method and the structure of DNA in stable transformants was examined. In most clones, the introduced DNA was integrated into the mouse genome in a tandemly repeated form. The possibility of multiple integration via mouse middle repetitive sequences was also examined using the chimeric plasmid with TK genes and middle repetitive sequences (pMRTK-1). Digestion with restriction enzymes showed that the middle repetitive sequence used in this experiment had no effect on the efficiency of transformation, suggesting that this sequence is unable to mediate homologous recombination with mouse genomes. |
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| Keywords: | Recombinant DNA plasmid vector pricking of nuclei tandem repeats of integrated DNA genetic transformation HAT selection HAT medium the medium containing hypoxanthine aminopterin and thymidine (Szybalska and Szybalski 1962) HSV-TK gene thymidine kinase deficient mouse L cell PBS phosphate buffered saline RSB reticulocyte standard buffer SDS sodium dodecylsulfate SSC |
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