[Construction of Zinc-alpha2-glycoprotein expression plasmid and its expression in 3t3-l1 preadipocytes].
OBJECTIVE
To construct mouse Zinc-alpha2-glycoprotein (mZAG) eucaryotic expression plasmid and identify its expression in 3T3-L1 preadipocytes.


METHODS
The total RNA from mouse liver tissue was extracted. The reverse-transcript(RT)-PCR method was used to amplify the complete domain sequence of mZAG, and the confirmed PCR products was inserted into expression plasmid by DNA ligation. The mZAG expression plasmids with various concentrations (0, 0.4, 0.8, and 1.6 microg) were transfected into 3T3-L1 preadipocytes, and ZAG expression in mRNA and protein level was determined by real-time fluorescence quantitative PCR and Western blot, respectively.


RESULTS
DNA sequencing confirmed the right sequence of mZAG expression plasmid pcDNA3.1(-)-mZAG. After the mZAG expression plasmid with different concentrations were transfected into 3T3-L1 preadipocytes, mZAG mRNA level significantly increased and reached 2.58 folds (P=0.002), 3.67 folds (P=0.000 and 5.19 folds (P=0.001) of that in the control group (no mZAG transfection). mZAG protein level also significantly increased and reached 2.75 folds of that in the control group (P=0.017). Treating 3T3-L1 cells with small interfering RNA (siRNA) sequence siRNA 1 and siRNA 4 resulted in a decrease of mZAG mRNA to 49% and 41% of those in the control group(no siRNA sequence transfection) (P=0.002P=0.000)and a decrease of mZAG protein to 55% and 62% of that in the control group (P=0.004,P=0.025).


CONCLUSIONS
mZAG expression plasmid pcDNA3.1(-)-mZAG was successfully established in this study. This plasmid can be well expressed in 3T3-L1 preadipocytes. siRNA 1 and siRNA 4 can effectively inhibit the expression of mZAG in these cells.
Zhang
Shao-Jun
SJ
Key Laboratory of Endocrinology of Ministry of Health, Department of Endocrinology, PUMC Hospital, CAMS and PUMC, Beijing 100730, China.
Gong
Feng-Ying
FY
Deng
Jie-Ying
JY
Zhu
Hui-Juan
HJ
Pan
Hui
H
Zhang
Dian-Xi
DX
Li
Nai-Shi
NS
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D005822:Genetic Vectors; D051379:Mice; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D029589:Seminal Plasma Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D000090203:Zn-Alpha-2-Glycoprotein
D034741:RNA, Small Interfering; D029589:Seminal Plasma Proteins; D000090203:Zn-Alpha-2-Glycoprotein