动物造模 z-bio 2021-11-30 315

　　Purpose: To screen and verify the effective silencing target of the marmoset B2m gene at the cellular level.

　　Method: Query the valid siRNA target site sequence verified by human B2m, compare the homology with the marmoset B2m gene sequence, and select the matching target to synthesize shRNA sequence. The two interference sequences synthesized in vitro were respectively connected with the lentiviral vector FUGW-TDT to construct a FUGW-TDT-shb2m interference expression plasmid, which was transfected into 293T cells under the mediation of polyethylenimine (PEI), 48h after transfection , Real-time fluorescence quantification method was used to detect the level of B2m mRNA in transfected cells.

　　Results: Two B2m silencing target sites that were completely homologous to marmosets were screened, which were located at 290-310 bp and 665-685 bp of B2m mRNA. The silencing efficiency of the two B2m targets at the transcriptional level was (46.54±). 7.91)% (P<0.05) and (83.22±4.37)% (P <0.0001), the difference is significant.

　　Conclusion: The FUGW-TDT-shb2m recombinant plasmid was successfully constructed; two effective B2m gene silencing targets were screened at the cell level; this laid the foundation for subsequent studies on mediating marmoset B2m gene silencing.