Establishment Of CD14Shrna Transgenic Mouse Model

Differentiation antigen14(also called CD14) is a high-affinity receptor protein of the complex of bacterial LPS, which can combine with LPS. CD14could partcipate in the digestion and phagocytosis of the Gram-negative bacteria to trigger the body’s immune responses. The purpose of present study was to explore the effects of expression inhibition of CD14on expression of related genes in vivo and individual development of transgenic animals, to establish transgenic mouse model.of anti-gram-negative bacteria. Screened and confirmed CD14shRNA fragment was used to construct its eukaryotic expression vector, transgenic mouse containing CD14shRNA were produced by pronuclear microinjection method. Considering the lower transgenic effciency of pronuclear microinjection process, some of influence factors of lentiviral-mediated transgenic techinique were also examined.1. Generation of transgenic mouse model of CD14shRNA by pronuclear microinjection.To construct the eukaryotic expression vector of CD14shRNA, screened and confirmed shRNA fagment of CD14gene was inserted into pSlience4.1-CMV neo vector. pSilencer4.1-CD14shRNA-IRES plasmid was injected into mouse by pronuclear microinjection to generate transgenic mouse.37founder pups were obtained, among them3were transgenic positive founder mouse by PCR analysis. Total33F1generation mouse were got, among them,11mouse were transgenic positive by the PCR and Southern-blotting check. To analyze the expression of exogenous genes, main organs of F1generation transgenic mice were checked by frozen section method. The results showed that the EGFP protein only expressed in liver, kidney and spleen, the expression in spleen was highest, which demonstrated that the obtained mouse were chimeric. The enogenous expression of CD14gene in F1transgenic mouse was detected by qRT-PCR method. The results showed that the expression of CD14gene in heart, liver, spleen, lung, kidney of transgenic mouse was lower than that of in control organs, decreased8-,3-,19.5-,6-and11times respectively. The results demonstrated that expression of CD14gene was inhibited in the obtained transgenic mouse.2. The effect of lentiviral vectors of CD14shRNA on mouse embryo development and transgenic efficiency.The effects of virus titer, injection period and U6promoter resource of lentiviral vector on mouse embryo development and transgenic efficiency were studied by perivtelline space injection method respectively. The results showed that the virus titer of1×106IU/mL～1×109IU/mL could infect the mouse embryos, there had no significant difference on the cleavage rates among the4groups(P>0.05). The blastocyst rate of1×109IU/mL group was significant lower than that of other groups, there was significant difference on the transgenic efficiency of EGFP gene among the4groups(P<0.05), The suitable titer for lentivirus infection of mouse embryos was1×108IU/mL. The lentivirus could infect the mouse embryos of1-cell and2-cell stages, there had no significant difference on the rates of cleavage, blastocyst and transgenic efficiency between the two stages(P>0.05), the2-cell injected groups were higher than that of1-cell groups, the appropriate injecton period was2-cell stage. Two lentiviral vectors with buffalo and human U6as promoter were used to infect1-cell stage mouse embyros, there had no significant difference on the rates of cleavage, blastocyst and transgenic efficiency between the two groups(P>0.05), transgenic rate of blastocyst of the human U6injected groups was higher than that of the buffalo U6groups (96.4%VS83.5%), EGFP expression in the human U6injected group was5.6times higher than that of the buffalo U6group, it indicated that the transgenic efficency of the human U6promoter was higher than that of the buffalo U6. The above obtained results would be used for next to generate transgenic mouse via lentiviral vector.Conclusions:CD14shRNA transgenic mouse model generated by pronuclear microinjection method could stably inherit the exogenous genes, the expression of enogenous CD14gene in transgenic mouse was significantly inhibited. Human U6promoter was better for lentiviral vectors of CD14shRNA, the suitable lentivirus infection period and titer for mouse embryos was2-cell and1×108IU/mL.