Silencing Effect Of Shrna With Different Stem Length On Egfp Gene Expression Of Transgenic Mouse

In this study, shRNA expression vectors with different stem length (21 bp, 27 bp, and 29 bp) were constructed, and the enhanced green fluorescent protein gene was used as target gene. Their silencing effect was tested in mouse cell and in vivo by cell transfection and pronuclear microinjection. The findings in this study are of interest for selecting the best hairpins for mouse individuals.1. Mouse embryonic fibroblasts were isolated and cultured from EGFP-transgenic mouse. Cells were more profuse and pure acquired from13.5 days embryonic age than other ages. It took about 3-4 days that the cell spreaded out the bottom of culture flask by trypsin digestion method, which was shorter than that (6-8 days) by tissue block method. Compared with other methods, it is time saving and has a high attachment rate that digested by 0.25% trypsin+ 0.02% EDTA for this cell passage. After several rounds of screening, DMEM(high glucose)+10% FBS was deemed to be the optimal culture medium, and 400μg/mL was considered the suitable selection concentration of G418 for EGFP -transgenic mouse embryonic fibroblasts.2. Aiming directly at the enhanced green fluorescent protein gene, shRNAs with different stem length (21 bp, 27 bp, and 29 bp) were designed and then ligated into psiSTRIKE expression vector. Target sequences were successfully cloned into the vector testified by enzyme digestion and sequencing.3. The recombinant plasmids were transfected into mouse embryonic fibroblast with lipofection and injected into leg muscle of mouse separately. The mRNA expression level of green fluorescent protein gene was checked by real-time fluorescent quantitative RT-PCR. EGFP-21siRNA, EGFP-27siRNA and EGFP-29siRNA regulated the gene expression down to 45%, 32%, 25%, respectively in cell. The silencing effect reached peak at 24h after transfection by EGFP-21siRNA and at 48h by EGFP-27siRNA or EGFP-29siRNA. EGFP-21siRNA, EGFP-27siRNA and EGFP-29 siRNA regulated the expression of target gene down to 8.9%, 9.7%, and 9.0%, respectively in vivo. There were no obvious difference among silencing effect of shRNAs with stem length of 21 bp, 27 bp and 29 bp. 4. Fluorescence labeled vectors of tandem shRNA with different stem length (21 bp, 27 bp, and 29 bp) targeting EGFP were successfully constructed. There was only light fluorescence observed under fluorescence microscope when these vectors were transfected into Vero cells. The mRNA expression level of green fluorescent protein gene was checked by real-time fluorescent quantitative RT-PCR. The results showed that the fluorescent expression of pGenesilR21, pGenesilR27, and pGenesilR29 was only 4.3%, 3.0% and 6.9% of the control group (pEGFP-C1) separately.5. The tandem shRNA expression vectors were injected into leg muscle of mouse. The mRNA expression level of green fluorescent protein gene was checked by real-time fluorescent quantitative RT-PCR. The results showed that fluorescent expression of pGenesilR21, pGenesilR27, and pGenesilR29 was only 2.5%, 1.3% and 5.1% of the control group (pEGFP-C1) separately.6. The interfering vectors were microinjected into male pronucleus of the embryos. When cultured in vitro, embryo developmental arrest took place in 2 cell stage. It was obvious that the fluorescence of microinjected embryos became weakened under microscope. 1192 of mouse fertilized eggs were microinjected altogether, and 763 of them survived after treatment in this study. Survival rate was about 64%. Only 23 of 44 transplanted female mice had pregnancies. There were 21 of mother mice aborting and only one giving birth to living offspring in the gestation period.7. The interfering vectors were injected into mouse tail vein in order to value their adverse effect. All of the mice did not die and had no adverse effect two months later after injection, while target gene had integrated in the mouse genome testified by PCR.8. 5 samples were positive in progeny of transgenic mouse detected by PCR method.