[Expression of green fluorescent protein vector by promoter sequence of CYP21 gene and CYP21P gene].
After amplification of PCR fragment of -770 bp(-)-1 bp in promoters of CYP21 gene and CYP21P gene respectively, the CMV promoter was cut off from pEGFP-N1, the vectors were constructed, in which contained promoter areas in CYP21 gene (pCYP21) and CYP21P gene (pCYP21P). All pCYP21, pCYP21P, pEGFP-N1(positive control) and negative control were transfected respectively into steroidogenic Y1 cell line, and were observed by inverted fluorescent microscopy and laser confocal microscopy. After transient transfection, the cells placed on inverted fluorescent microscopy. The appearance of GFP expression observed is as follows: pEGFP-N1 at 3 hours; pCYP21 at 7 hours. However, neither pCYP21P nor negative control expressed GFP. Laser confocal microscopy showed that pEGFP-N1 and pCYP21 produced GFP. pEGFP-N1 is stronger than pCYP21, but none of pCYP21P and negative control expressed GFP. Different distribution of GFP in Y1 cell could be seen of pEGFP-N1 and pCYP21, and the intensity of GFP in nucleus is stronger than cytoplasm. Our results further conform that there is a significantly difference of GFP expression in Y1 cell line by promoters of CYP21 and CYP21P.
Han
Bei
B
Xinhua Hospital SSMU Shanghai 200092, China.
Wang
Xiu-Min
XM
Zhang
Ya-Fen
YF
Gu
Xue-Fan
XF
D000818:Animals; D001483:Base Sequence; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011544:Pseudogenes / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013255:Steroid 21-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013255:Steroid 21-Hydroxylase