Establishment And Phenotype Analysis Of Trim30a Knockout Mouse Model

TRIM30??Tripartite motif protein 30??is a member of the TRIM protein family,plays an important negative regulatory role in the innate immune system.Studies have shown that TRIM30?can negatively regulate DNA virus-mediated immune response through multiple immune signaling pathways,but whether there are other modes of regulation remains unclear,and what associations exist between several known modes of regulation remains to be further elucidated.So we created Trim30a knockout Chimera mice using the CRISPR/CAS9 technique,the Trim30a Gene knock-out homozygous mice(Trim30a^-/-)were obtained by genetic breeding and a series of phenotypic analysis experiments were carried out to determine whether our Trim30a gene knock-out mouse model was successful or not,this study will lay a foundation for further research on the role of TRIM30 in innate immune response.In this study,the first segment of mouse Trim30a gene was used as the target sequence to knock out.Two pairs of SGRNA were designed using this sequence as the template,and were linked and transformed with p UC19-T7 plasmid,two recombinant plasmids containing SGRNA?p UC19-T7-Trim30a-sg RNA1,p UC19-T7-Trim30a-sg RNA2?were constructed and identified by sequencing.Two recombinant plasmids were transcribed in vitro,and the transcribed products of T7-Sp Cas9-SV40 plasmids were injected into mouse embryonic cells at a molar ratio of 1:1,the chimeric mice?F0?with Trim30a gene knock-out were obtained by implanting the chimeric mice.The TRIM30a^+/-mice?F1?were bred from F0 generation knock-out mice and WT mice,and then F2 generation mice were bred in full-sib cage.Seven Trim30a^-/-mice were screened by PCR and sequencing,and the phenotype of homozygotic mice was analyzed.The knockdown of Trim30a genes in mouse genome was identified by PCR amplification and non-denaturing polyacrylamide gel?PAGE?electrophoresis;RT-PCR detection the expression of Trim30a gene RNA in mice was measured;Off-target effect was detected by T7E1 restriction enzyme digestion;the body weight of mice was measured at 3?8 weeks,and the effect of knockout on the growth and development of mice was observed;detection of mouse blood index analysis Trim30a^-/-mouse metabolic indicators and blood cell production abnormal.The results showed that the Trim30a gene was deleted in the major tissues and organs of Trim30a^-/-mice,the Trim30a m RNA of homozygous mice was not detected by RT-PCR;Off-target editing was not detected in homozygous mice Compared with wild-type mice;homozygous mice raised under normal conditions had no significant difference in appearance,development and metabolic indexes;the blood test showed that Trim30a^-/-mice had no significant difference in all indexes compared with WT mice.To sum up,the Trim30a gene was successfully knocked out by CRISPR/Cas9 technique and seven Trim30a^-/-mice were obtained after genetic breeding.The phenotypic analysis showed that Trim30a gene of Trim30a^-/-mice had been systematically knocked out and not expressed.The deletion of Trim30a gene in homozygous mice could be stably inherited to the next generation,and there was no significant difference between the physiological and biochemical indexes of Trim30a^-/-mice and the WT mice of the same strain.Above results indicate that Trim30a^-/-mouse model is constructed successfully,providing experimental materials and research basis for further elaboration of the role played by TRIM30?in DNA virus-mediated innate immune response.