小鼠4.5S RNA逆转座子对Luc基因表达的影响

EFFECT OF MOUSE 4.5S RNA RETROTRANSPOSONS ON THE EXPRESSION OF Luc GENE

利用RT PCR方法 ,从小鼠肝脏组织总RNA中扩增出 4 5SRNA的cDNA。该cDNA被克隆到pGEM3Zf ( + )质粒上 ,酶切鉴定并测序。然后将该序列插入以Luc基因作为报道基因的表达载体pSVluc2 0的PvuⅡ位点 ,构建了含 4 5SRNA逆转座子的表达载体pSVluc2 0 4 5S。脂质体转染法将表达载体导入小鼠骨髓瘤细胞NS 1、SP2 /0和人乳腺癌细胞Bca61。结果表明 ,小鼠 4 5SRNA逆转座子序列在NS 1、SP2 /0细胞中对Luc基因的表达有促进作用 ,而在Bca61细胞中对报道基因的表达则起抑制作用。

The cDNA of 4 5S RNA was amplified by reverse transcription polymerase chain reaction (RT PCR) method using total RNA extracted from Kunming mouse's liver tissue as the template. The cDNA fragment was inserted into pGEM3Zf(+) plasmid, restriction enzyme digestion detected and sequenced. The 4 5S gene sequences of Kunming mouse, the mouse (Harada et al., 1980) and the hamster (Harada et al., 1980) were studied and found that there are a few different nucleotides in the 88 base pair, and the different nucleotides were not involved in 4 5S gene repeat regions. Then the 4 5S gene was inserted into the upper of SV40 promotor in vector pSVluc20 and constructed the expression vectors, pSVluc20 4 5S(+) and pSVluc20 4 5S(-). And these vectors were transferred into mouse's NS 1, SP2/0 cells and human Bca61 cells by liposome transfection experiments. These transfer cells were continually cultured 48 hours, and then the cellular luciferase activity were detected. The results showed that after 4 5S gene was inserted into the vector by different direction, no difference was produced in the Luc gene expression. The activity expression of Luc gene of the vectors for constructing mouse's 4 5S gene in cultural cells NS 1 and SP2/0 was two times as much as the contrast pSVluc20. The Luc gene of expression vectors could not expressed in the Bca61, but the contrast was normally expressed. In addition, there was a marked drop in the activity of luciferase as the culture time of transferred cells were prolonged. Falling speed of enzyme activity in the experiment groups was larger compared with the contrasts.