Fertilized rabbit eggs injected with SMTPGH gene were cultured in vitro and retention of theinjected gene was studied using PCR technique and nonradioactive labelling methed. In a mediumof TC199 + 10% FCS, three quart...Fertilized rabbit eggs injected with SMTPGH gene were cultured in vitro and retention of theinjected gene was studied using PCR technique and nonradioactive labelling methed. In a mediumof TC199 + 10% FCS, three quarters of the fertilized eggs developed to the blastocyst stage. Noapparent change of the injected gene was found before the 8-cell stage, after which it was eitherintegrated into the chromosome of the host or lost gradually. But finally, the retention rate of theinjected gene should be equal to its integration rate.展开更多
An expressive plasmid pSMTPCH was constructed from porcine growth hormone gene,sheep metallothionein promoter (MT-011)and the vector,pUC19. The linear pSMTPGH and circular pSV2-dhfr were cotransfected into CHO-dhfr ce...An expressive plasmid pSMTPCH was constructed from porcine growth hormone gene,sheep metallothionein promoter (MT-011)and the vector,pUC19. The linear pSMTPGH and circular pSV2-dhfr were cotransfected into CHO-dhfr cell by calcium phosphate coprecipitation. Positive clones made up 74% of total clones, which were identified with ELISA. The expression of pSMTPGH was induced by 0.5 μM of Cd￣++. The clone 1-C-3 was found to secrete hGH at the level of 3800 μg/10￣6 cells/24 hrs in media containing 10 μMTX. After 20 generations in culture, the clone was still stable with hGH expression.The molecular weight of secreted protein was the same as that of the natural pGH, 22KD;the identity was further supported by Western blot.展开更多
基金This work was supported by National High Technoloqy Grants 86310105.
文摘Fertilized rabbit eggs injected with SMTPGH gene were cultured in vitro and retention of theinjected gene was studied using PCR technique and nonradioactive labelling methed. In a mediumof TC199 + 10% FCS, three quarters of the fertilized eggs developed to the blastocyst stage. Noapparent change of the injected gene was found before the 8-cell stage, after which it was eitherintegrated into the chromosome of the host or lost gradually. But finally, the retention rate of theinjected gene should be equal to its integration rate.
文摘An expressive plasmid pSMTPCH was constructed from porcine growth hormone gene,sheep metallothionein promoter (MT-011)and the vector,pUC19. The linear pSMTPGH and circular pSV2-dhfr were cotransfected into CHO-dhfr cell by calcium phosphate coprecipitation. Positive clones made up 74% of total clones, which were identified with ELISA. The expression of pSMTPGH was induced by 0.5 μM of Cd￣++. The clone 1-C-3 was found to secrete hGH at the level of 3800 μg/10￣6 cells/24 hrs in media containing 10 μMTX. After 20 generations in culture, the clone was still stable with hGH expression.The molecular weight of secreted protein was the same as that of the natural pGH, 22KD;the identity was further supported by Western blot.
