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사람 HPRT 유전자 발현벡터 제조와 생쥐 Sp2/0 myeloma 세포주에서의 발현
고창보,김용만,김영진,백상기 충남대학교 기초과학연구소 1997 忠南科學硏究誌 Vol.24 No.1
To construct expression vector for the human HPRT gene, pRSVneo plasmid conferring resistance to neomycin-kanamycin(Tn5) antibiotics and pHPT 31 containing human HPRT cDNA were used to subclone human HPRT cDNA. For construction of recombinant pRSVneo carrying human HPRT, pRSVneo-HPRT, the human HPRT cDNA fragment from pHPT31 plasmid was inserted into polyadenylation site (BamHI site) behind the neo gene fragment of pRSVneo plasmid. For the other expression vector, pRSV-HPRT, the neo gene and small portion of the untranslated 5′franking region were removed from the pRSVneo-HPRT. The HPRT gene of the two vectors were constructed in right orientation. Each recombinant vector was introduced into cultured HPRT-deficient mouse cell line, Sp2/0 by calcium mediated DNA transfection and the transfected cells were selected under HAT selection condition. The HPRT activity of the lysates from the selected cell was higher than that of the lysates from spleen cells of mouse. The HPRT activity of pRSVneo-HPRT vector was higher than that of pRSV-HPRT one. To find out whether the HPRT activity of the transfected cells selected under HAT medium was expressed by transfected vectors or by spontaneous mutations, it was cultured to reselect in G418 medium. It showed that the nature of the HPRT activity was resulted from the transfected vectors.