PROMOTER FUNCTIONAL
ANALYSIS OF CYP21B AND CYP21A BY GREEN FLUORESCENT PROTEIN
Han B, Wang XM, Gu XF
Department of Research
Center, Xin Hua Hospital, Shanghai Second Medical University, Shanghai, China
Objective: About 95% of congenital
adrenal hyperplasia (CAH) are caused by 21-hydroxylase deficiency. We use
green fluorescent protein plasmid to analysis function in promoters of
CYP21 B and CYP21A gene.
Methods: specific PCR was used to
amplify fragments of promoter area of CYP21 B and CYP21A gene. Two pEGFP-N1
plasmids were constructed including promoter of CYP21 B and CYP21A gene,
which confirmed by enzyme digestion and DNA sequencing. Reporter plasmids
were transfected into Y1 cell line and cos-1 cell line transiently.
Different groups were observed under confocal laser microscopy, and
fluorescent microscopy. Fluorescent intensity of different groups was
detected by flow cytometry.
Results: There are obvious
differences in 4 groups, the fluorescent intensity was following: negative
control and Pegfp-N1-CYP21A < Pegfp - N1 - CYP21B < Pegfp - N1
(positive control). Flow cytometry and other quantity methods also
confirmed it.
Conclusions: Promoter function in
CYP21A is lower than CYP21B in pEGFP-N1 vectors. It is a new way to explore
promoter function of 21-hydroxylase.