[Expression of mutation type GJA8 gene and wild type GJA8 gene of a congenital inherited nuclear cataract family in eukaryotic cells].
OBJECTIVE
To clone the sequence of mutation type GJA8 gene (mGJA8) and wild type GJA8 gene (wGJA8) of a congenital inherited nuclear cataract family and study their expression in eukaryotic cell lines in vitro.


METHODS
The mGJA8 and wGJA8 were amplified from this family's DNA and healthy people's DNA by PCR respectively. The mGJA8 and wGJA8 were recombined with plasmid pEGFP-N1 respectively. The accuracy of pEGFP-N1-GJA8 was confirmed by restriction enzyme digestion and DNA sequencing. Finally pEGFP-N1- mGJA8 and pEGFP-N1- wGJA8 and GFP protein were transfected into COS7 cells by lipofectin. The expression of pEGFP-N1-GJA8 and GFP fusion protein were to observe under fluorescence microscope, and to detect by Western-blotting and immunohistochemical staining.


RESULTS
The mGJA8 and wGJA8 were cloned successfully. With restricting enzyme digestion analysis and DNA sequencing, recombinant plasmid pEGFP-N1-mGJA8 and pEGFP-N1-wGJA8 were constructed correctly and their GFP fusions were expressed in transfected COS7 cells. The expression of pEGFP-N1-mGJA8 and pEGFP-N1-wGJA8 fusion protein were observed under fluorescence microscope, and detected by Western-blotting and immunohistochemical staining successfully.


CONCLUSIONS
The mGJA8 gene and wGJA8 gene are cloned successfully, and pEGFP-N1-mGJA8 and pEGFP-N1-mGJA8 fusion protein can be expressed in COS7 cells, which establish the foundation for further studying the mechanism of this congenital inherited nuclear cataract family.
Zheng
Jian-qiu
JQ
Eye Hospital, First Affiliated Hosptial of Harbin Medical University, Harbin, China. zjq751130@sina.com
Liu
Ping
P
Wang
Jian-wen
JW
Liu
Jian-ju
JJ
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017630:Connexins / Q000096:biosynthesis* / Q000235:genetics*; D005136:Eye Proteins / Q000096:biosynthesis* / Q000235:genetics*; D015316:Genetic Therapy; D005838:Genotype; D006801:Humans; D009154:Mutation; D009679:Nuclear Family; D010375:Pedigree; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D000099264:Connexin 50
D017630:Connexins; D005136:Eye Proteins; D011993:Recombinant Fusion Proteins; D000099264:Connexin 50