Experimental Study On The Recombinant Pc-DNA3-NT-4 Transfecting BMSCs Of GFP Transgenic Mice

Objective To construct the eukaryotic expression vector of rat neurotrophin-4(NT-4) total gene and express it in BMSCs of GFP transgenic mice.Methods The gene of rat neurotrophin-4(NT-4) was amplified by RT-PCR from hippocamp tissue. By gene recombination technique, rat NT-4 cDNA was inserted into mammalian expression vector pcDNA3. The recombinant plasmid vector which contains the inserted sequence was verified with restriction enzyme digestion analysis and DNA sequencing. BMSCs were transfected with this vector using Lipofectamin 2000 transfection reagent. After transfection, the transfected cells were grown in DMEM medium containing G418, and the positive clones were selected in DMEM medium until the 30^th day. The expression of NT-4 was analyzed by immunocytochemistery and the biological activity was analyzed by the neurite outgrowth of PC12 cells stimulated by culture supernatant of positive clone.Results Total RNA showed 18S and 28S two bands in electrochromatography,RT-PCR product in the agarose gel elctrophrosis show that there had positive strap,which matched the expected size.Through digested and sequenced,the inserted DNA sequence was the gene of NT-4. After transfected the recombinant vector, the PC12 cells increased in the NT-4-containing conditioned medium and stretched out long neuritis,as well as compared with the vector control...