动物造模 z-bio 2022-06-07 381

　　OBJECTIVE: To screen and verify the effective silencing target of 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 site to synthesize shRNA sequence. The two interfering sequences synthesized in vitro were respectively connected with the lentiviral vector FUGW-TDT to construct a FUGW-TDT-shb2m interfering expression plasmid, which was transfected into 293T cells under the mediation of polyethylenimine (PEI), 48h after transfection , the level of B2m gene mRNA in transfected cells was detected by real-time fluorescence quantitative method.

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

　　Conclusion: The FUGW-TDT-shb2m recombinant plasmid was successfully constructed; two effective B2m gene silencing targets were screened at the cellular level, which laid the foundation for the subsequent research on the mediation of B2m gene silencing in marmosets.