Production Of Albino C57BL/6N Mice And ICSI Of Dead Mouse Sperm

In this study,C57BL/6N mice were used as the research object.The albino C57BL/6N mice were made by CRISPR-Cas9 technology and the transgenic strains were restored by sperm of dead mice.In order to obtain albino C57BL/6N mice,the CRISPR-Cas9 technology was used and the Cas9 mRNA and a series of sgRNAs were designed and synthesized which were injected into fertilized eggs.And the tyrosinase(TYR)gene of C57BL/6N mice was destroyed to generate gene mutant mice,by repeated backcrossing and selfing,formed C57BL/6N albino mouse inbreds.The results show:after injection of two pairs of sgRNA,F0 generation albino mice were successfully obtained.Deletion mutations were occurred in the first and second exons of Tyr gene,and the first exon was deleted by 45 bp and 14 bp,the second exon was deleted by 7 bp and 3 bp.The mouse can transmit the mutant gene to the offspring and homozygous white C57BL/6N mice were produced in its offspring.The C57BL/6N albino mouse inbred line was successfully established,which will provide new research tools for future chimera preparation and tissue transplantation.To investigate whether the sperm of the dead transgenic mouse strain has the ability to activate the oocyte and allow the embryo to continue to develop to produce pups,the Nestin-GFP transgenic male mice were killed to collect sperm after 14 h,24 h,48 h of death,and sperms were injected into C57BL/6N and B6D2F1 mouse oocytes respectively,after developing to 2-cell,embryos were transplanted into surrogate receptors,and some B6D2F1 derived embryos were observed and cultured in vitro.At the same time,the histone H2 AX phosphorylation of B6D2F1 embryonic parternal pronucleus was detected by immunofluorescence technique.The results show:the dead sperms of the three experimental groups were all can activate and allow the C57BL/6N and B6D2F1 oocytes fertilize,and the F0 generation mice were successfully obtained after 2-cell embryos were transplanted.Also the F0 mice had normal reproductive ability,and the F1 generation mice were successfully obtained.At the same time,the characteristic of the transgene was preserved and inherited.The ICSI embryos derived from B6D2F1 were developed in vitro: the cleavage rate and blastocyst rate of the three experimental groups were significantly lower than those of the control group(0 h).The birth rates of B6D2F1-derived ICSI embryos at 24 h and 48 h were significantly lower than those in the 14 h group and the control group(0 h).The cleavage rate of C57BL/6N-derived ICSI embryos at 24 h and 48 h were significantly lower than that of the 14 h group and the control group(0 h),and the birth rates of the three experimental groups were significantly lower than the control group(0 h).Phosphorylation assay of histone H2 AX showed that the proportion of non-focus embryos in the three experimental groups were extremely significant lower than that in the control group(0 h);the proportion of non-focus embryos at 48 h was extremely significant lower than 14 h and 24 h;After the Wilcoxon’s test,the number of foci in the three experimental groups were extremely significant higher than that in the control group(0 h).In summary,by use of ICSI technology,the sperm of the dead mouse at 14 h,24 h,48 h after death is capable of activating the oocyte and allowing the embryo to continue to develop until the pup is born.This study provides an effective way to rescue accidentally dead transgenic mouse strains.