Preliminary Study On Generation Of Micro Mice And Micro Tibetan Minipig Using CRISPR/Cas9 Gene Modification Technique

Background and objectiveCurrently,the minipigs are becoming the most widely used large laboratory an-imals due to their short stature,easy operating,and sharing anatomical,physiological and biochemical similarities to humans.The Tibetan minipig is a great animal model,while with large size,high cost of feeding and difficulty in keeping.The micromation of Tibetan minipigs,ensuring that organ function remains the same but anatomy scaled down,can reduce the breeding cost and research cost and simplify the experi-mental operation.Furthermore,it is of great significance to breeding SPF miniature Pig.In the regulation of animal growth,GH-GHR-IGF1 signal transduction pathway plays an important role.GH combines with GHR on the surface of the target cell to promote the synthesis of IGF1,which is important to promote the growth and prolif-eration of cells.The GHR gene,located in chromosome 16 of the pig,is composed of 10 exons and widely expresses in all tissues and organs.The mature GHR protein in-cludes extracellular domain(growth hormone binding domain),transmembrane do-main and intracellular domain.The mutations in GHR genes are closely associated with human and animal dwarfism,and the mutations in different structural domains may result in small size[1-3].IGF1 is the downstream gene of GHR,which is a product through stimulation of liver by GH.IGF1 can promote the growth of body cells through endocrine,paracrine and self-secretion approach.It has been reported that the lack of GHR or IGF1 or both interfere with the growth and development of individuals,resulting in slow growth and small size.Against this background,we will intend to generate small but normal Tibetan miniature pigs via CRISPR/Cas9 technology by interference with GH-GHR-IGF 1 signal transduction pathway.Research contents and methodsThe study intend to obtain micro Tibetan minipigs using the CRISPR/Cas9 sys-tem,a recently developed and mature gene editing techniques,tageting GHR and IGF1 gene.The pig with modification of GHR/IGF1 could be applied to the study of the process of the role of GHR and IGF1 and the research of dwarfism or act as a smaller animal model in basic researches.There is no study of the success of the IGF1 gene knockout in pigs reported,and the generation of gene-editing pigs is time-consuming,so we choose first to knock out IGF1 in mice using the CRISPR/Cas9 system.ResultsIn this study,We successfully obtained IGF1 homozygous knockout and hetero?zygous knockout mice.A total of seven FO generation mice were produced,among which one mouse was wild-type,and the others were mutated mice at the sgRNA site,with the knockout efficiency of 85.71%.The homozygous knockout mouse weighed only 4.8g(-30%of the weight of WT mice)at 4 weeks;Anatomy and histological examination showed that the size of organs were significantly reduced with no obvi-ous abnormality.IGF1 gene expression was down regulated.There was no difference in weight of heterozygous knockout mice and wild-type miceWe knocked out the exon 2 in Tibetan minipigs using the CRISPR/Cas9 system,and finally four FO generation piglets were obtained.Among these pigs,two pigs were heterozygotes knockout,and the others were wild-type.The weight,body length and body height of two heterozygous knockout pigs were smaller compared with those of wild-type pigs.The expression of GHR in mutated pigs were down regulated in both mRNA and protein level.Compared with the wild type,the serum IGF1 level in the mutated pigs was decreased,with statistical significance,P<0.05.No signifi-cant differences were obsearved in insulin and blood glucose levels.No off-target ef-fect of CRISPR/Cas9 system in the exon2 of GHR was detected.In this study,the GHR-knockout pigs did not show the same micro phenotype as GHR-deficient pigs in other studies,partly because of:1.Most of the obtained zygotes were in 2-cell stage,and Cas 9 did not fully function in all single cells;2.Mutations caused by sgRNA targeting GHR gene did not result in complete afunction.ConclusionWe obtained heterozygous GHR-knockout Tibetan minipigs and IGF 1-knockout mice.The generation of homozygous IGF 1-knockout mouse suggests that the targrt of IGF1-E2 is effective,and can be applied to knock out the appropriate site of IGF1 gene in pigs to obtain micro Tibetan minipigs.The Tibetan minipigs with heterozy-gous GHR-knockout can be mated and bred to obtain the GHR-knockout homozy-gotes...