Study On The Generation Of Albino And Immunodeficient Tibet Minipigs By Zygote Injection Of CRISPR/Cas9

Being a rare breed of plateau pig breeds,Tibet minipig demonstrates its fitness for animal experiment with characteristics of slow growth,small size,well-proportioned shape,strong resistance to disease and stress,and good tolerance to surgery.However,Tibet minipig’s dark black fur color posed quite an inconvenience to experimental execution,in contrast to popularity of laboratory animals of albino strains with researchers,for which reason albino Tibet Minipig will be desirable for more extensive medical application.Practically,breeding immunodeficient Tibet minipigs will be also invaluable in biomedicine,examples of which can be such as preparation of human organs,reconstruction of human immune system,and xenogeneic stem cell transplantation.The presented study explored and established an optimized strategy and reliable system of preparation of albino and immunodeficient gene-modified Tibet minipigs by CRISPR/Cas9 zygote cytoplasmic injection method.To outline the key steps,firstly,estrus in Tibet minipigs was rendered among selected Tibet minipigs by altrenogest and the optimum mating timepoint was determined by the developed vaginal smear method.These critical steps help us procure an efficient and stable collection of naturally fertilized 1-cell stage embryos.Also,to verify and set up a sound technical system,albino mice of TYR-knockout(tyrosinase gene related to melanin synthesis)were generated through cytoplasmic injection of zygotes with CRISPR/Cas9.Based on these,Tibet minipigs’ tyrosinase gene(TYR),interleukin-2 receptor gamma subunit gene(IL2RG)related to immunity and recombinant activator gene 1(RAG1)were selected as target genes,which were edited by CRISPR/cas9 gene editing technology.One or two sgRNAs were designed for each target gene.To be noted,for obtainment of the homozygous knockout albino individuals,single TYR gene was edited in 1-cell stage embryos,while for obtainment of albino and immunodeficient dual gene-edited individuals or corresponding chimeras,TYR-IL2RG or TYR-RAG1 dual genes were edited in 2-cell stage embryos.In summary,we successfully obtained albino(TYR-/-)Tibet minipigs,albino and immunodeficient Tibet minipigs(TYR-/-IL2RG-/-and TYR-/-RAG1-/-and TYR or IL2RG chimeras respectively.Genotyping of F0 generation piglets showed the overall gene editing efficiency was 75%for the TYR single gene knockout,while for TYR-IL2RG and TYR-RAG1 dual gene editing,the values were 25%and 75%,respectively.The gene-edited albino Tibet minipigs were further bred and its population expanded.No significant physiological difference in body size,reproductive performance,blood physiological and biochemical indexes were observed between albino Tibet minipigs and wild type Tibet minipigs(P>0.05).Also we have prepared IL2RG chimeras by 2-cell stage embryos editing,and the mutated IL2RG gene can be passed on from the F0 minipigs to the next generation by germ line transmission.In conclusion,we have successfully prepared TYR knockout albino Tibet minipigs by CRISPR/Cas9 zygote cytoplasmic injection and carried out population expansion and breeding research;in addition to obtainment of IL2RG knockout or RAG1 knockout immunodeficient Tibet minipigs,we proposed and verified the strategy of preparing chimeras of IL2RG or RAG1 genes by zygote cytoplasmic injection at the 2-cell stage embryos for purpose of breeding and seed preservation of immunodeficient Tibet minipigs.Based on this long-term project,a whole set of mature technical system has been established as described above,providing a vaild guidance for the efficient generation of gene-modified Tibet minipigs.