[Cloning and biological regulating effects of human WAF1 gene].
OBJECTIVE
To clone human WAF1 gene and construct WAF1-pcDNA3 recombinant plasmid, and to explore the biological regulating effects of WAF1 gene.


METHODS
Human WAF1 gene was amplified and cloned into pcDNA3 vector by RT-PCR and DNA direct sequencing from Hela cells. By immunofluorescence and immunohistochemistry staining, the identify the expression of the inserted WAF1 gene and its biological regulating effects on Rb, cyclin D1 gene.


RESULTS
Human WAF1-pcDNA3 recombinant plasmid was correctly constructed and transfected into Hct-8 cells. P21WAFI/Clip was successfully expressed. The functional characterization of the inserting WAF1 gene implied that P21WAF1/Cip1 expression could upregulate Rb gene and downregulate cyclin D1 gene at protein level.


CONCLUSIONS
Human WAF1-pcDNA3 can modulate the protein expression of the downstream gene.
Hou
Z
Z
Third Clinical College and School of Basic Medical Sciences, Norman Bethune University of Medical Sciences, Changchun 130031, China.
Bu
L
L
Wang
H
H
D003001:Cloning, Molecular; D019938:Cyclin D1 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics* / Q000502:physiology*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015536:Down-Regulation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016160:Retinoblastoma Protein / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D018076:DNA, Complementary; D016160:Retinoblastoma Protein; D019938:Cyclin D1