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| Conditional Expression Vector of Pig Growth Hormone Leads to Regulatable Expression in PK15 Cells by Tetracycline(TET-ON) System |
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Abstract Doxycyline(DOX)-inducible expression systems provide strict control over transgene expression spatially as well as temporally to avoid non-specific expression. Moreover DOX has no cytotoxic effects to the cells and animals, so it is used widely in transgenic research. Ptet-on system is more sensitive to DOX and yields lower background expression than that of the other inducible systems. For realizing the controllable expression of exogenous growth hormone (GH) gene at a specific time and space, we constructed inducible expression vector about tetracycline (TET-ON) system, and did a further study for testing its inducible activity at cellular level. The results showed that: (1) we have successfully constructed vector about pTRE-GH12; (2) we have harvested GH cells which had high inducible expression efficiency; (3)after induced by using DOX in the positive cells, we could get detection of the expression of exogenous GH, QRT-PCR results showed that the induction activity after using DOX was 264.481 times as before; there were no change in control group cells using or no using DOX , the differences between experimental group and control group were significantly different (P<0.01), so as the induction of DOX before and after in experimental group (P<0.01); (4)the results of Western blot were consisted with the result of QRT-PCR , the quantity of GH protein between the experimental group and control group cell showed significant differences (P<0.01), so as the experimental group before and after using DOX (P<0.01). All of our research results indicated: we have realized controllable expression of targeted gene pig growth hormone (pGH) by using tetracycline (TET-ON) inducible expression system, so as to provide a technological basis for preparing transgenic animals which can express GH inducible later.
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Received: 10 July 2012
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