[Construction of eukaryotic expression vector containing hSNCA gene and its pathogenic mutants and its expression in PC12 cells].
OBJECTIVE
To construct recombinant eukaryotic expression vectors pEGFP-C3-SNCA containing human wild-type (WT) and pathogenic mutations A30P, A53T alpha-synuclein (SNCA), and to obtain monoclonal PC12 cell lines overexpressing human wild-type and pathogenic mutations A30P, A53T alpha-synuclein by stable transfection.


METHODS
Human wild type SNCA gene was cloned by using RT-PCR. By T-A extension cloning, the gene was ligated with T-vector and sequenced. Based on it, the recombinant eukaryotic expression vectors containing wild type SNCA synonymous mutation or its G88C (Ala30Pro) and G209A (Ala53Thr) pathogenic mutation were constructed by site-directed mutagensis using primer variance in mononucleotide. And different recombinant plasmids pEGFP-C3-SNCA were identified with PCR, restriction enzyme digestion and DNA sequencing. PC12 cells were transfected with different recombinant plasmids pEGFP-C3-SNCA by liposome transfection method, screened with G418, and subcloned by limited dilution method to obtain different monoclonal PC12 cell lines stably over-expressing human wild-type, A30P or A53T alpha-synuclein, respectively, and PC12 cells stably transfected with pEGFP-C3 were used as control group. These monoclonal PC12 cell lines were identified with RT-PCR, Western blot and fluorescence microscopy.


RESULTS
According to result of PCR, the double digestion and gene sequencing, it was comfirmed that recombinant eukaryotic expression vectors containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutation had been constructed successfully. By means of RT-PCR, Western blot and fluorescence microscope, it was comfirmed that objective gene sequences in PC12 cells were stably over-expressed.


CONCLUSIONS
The recombinant pEGFP-C3-SNCA vectors containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutations had been constructed successfully. The transfected monoclonal PC12 cells are obtained in which three SNCA gene isoforms had stable expression.
Qian
Jin-jun
JJ
Department of Neurology, Second Affiliated Hospital of Soochow University, Suzhou 215004, China.
Cheng
Yan-bo
YB
Liu
Chun-feng
CF
Yang
Ya-ping
YP
Liu
Kang-yong
KY
D000818:Animals; D000942:Antigens, Bacterial; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D056890:Eukaryota / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D016716:PC12 Cells; D010957:Plasmids; D051381:Rats; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051843:Synucleins; D014162:Transfection; D051844:alpha-Synuclein
D000942:Antigens, Bacterial; D011993:Recombinant Fusion Proteins; D051843:Synucleins; D051844:alpha-Synuclein