大鼠肝脏细胞核白蛋白基因转录活性测定

THE ASSAY OF IN VITRO TRANSCRIPTION IN ISOLATED NUCLEI FROM RAT LIVER

目的：探讨在有限实验条件下，应用游离细胞核基因转录活性测定方法的可能性。方法：采用三因素、二水平正交设计表。结果：对大鼠肝脏白蛋白基因转录活性的最适测定条件为：ｃＤＮＡ上样量２μｇ；细胞核与底物孵育最适温度３０℃；［α－３２Ｐ］－ＵＴＰ的使用量１４８０ｋＢｑ。结论：应用此实验条件可使放射性核素及ｃＤＮＡ探针用量减少，降低核基因转录活性实验成本，提高其应用可行性。

Objective:To assess the appropriate condition of assay for in vitro albumin gene transcription in isolated nuclei from rat liver. Methods: Orthogonal experiment design[L(23)] was supplied as fac-tors of amount of[α-32P]-UTP(1 480 kBq, 2 960 kBq),albumin cDNA(2μg,4μg),and temperature of incubation for experiment(30 ℃, 20 ℃) at two levels,Results:The best conditions are 1 480 kBq of[α-32P]-UTP,2 μg albumin cDNA probe and incubation at 30 ℃ Conclusion:In these condition,nuclear run-on assay will be available for specific study because it hecomes much cheaper than it usually does.