Construction Of IGF-1 Class ? Splicinge Isoform Gene Knockout Rabbits

Insulin-like growth factor-1(IGF-1)is a multifunctional cell proliferation regulator.IGF-1 contains six exons,IGF-1 formed a variety of different mRN A splicing isoforms because of alternative splicing of different initiation sites and termination sites.According to different transcriptional initiation sites,IGF-1 can be divided into two types: class I transcripts(initiation sites located o n exon 1)and class ? transcripts(initiation sites located on exon 2).IGF-1 possesses a variety of biological functions,including that(1)IGF-1 regulates body growth and development together with growth hormone(GH)through GH/IGF-1 axis;(2)IGF-1 has a direct promoting effect on bone gr owth.It can promote osteoblast development and proliferation,the growth of l ong bone and increased bone density;(3)IGF-1 plays an important role in c ontrolling carbohydrate,protein,lipid and mineral metabolism.It can affect th e distribution of human body composition,including muscle specific gravity,f at,and body weight;(4)GH/IGF-1 axis can slow down the decline in animal cognitive ability and extend the life span of many species.Researchhave shown that various biological functions of IGF-1 are insepa rable from its multiple alternative splicing isoforms.Different splicing isoform s have different functions on orginisms growth and development,IGF-1 Class I splicing isoforms are more important in organisms,accounting for more th an 70% of the total IGF-1,while the proportion of IGF-1 Class ? splicing is oforms is only 20% or more.In this experiment,the IGF-1 Class I splicing isoform gene in rabbits we re targeted by using CRISPR/Cas9 gene editing technology,and IGF-1 Class I splicing isoform gene knockout rabbits were obtained by Cas9/sgRNA injecti on.The results showed that,compared with normal rabbits,IGF-1 knockout r abbits exhibited no mRNA expression of IGF-1 class I splicing isoforms and nearly unchangable mRNA expression level of IGF-1 class ? splicing isoform s.In addition,the protein expression level of IGF-1 was significantly reduced.At the same time,the phenotypes of the IGF-1 class I splicing isoform gene knockout rabbits was analyzed.The results showed that the gene knockout r abbits exhibited characteristics of growth retardation and smaller individuals in comparsion with the normal rabbits.Furthermore,gene knockout rabbits sho wed abnormal glucose and lipid metabolism and muscle fiber thinning.In summary,in this study,we successfully constructed IGF-1 class I spli cing isoform gene knockout rabbits with CRISPR/Cas9 technology.This novel rabbit model will provide theoretical basis for studying different biological fu nctions of different IGF-1 alternative splicing iosforms in animal orginisms.