Targeted Knockout Of Mouse MSTN Gene By Engineered Zinc Finger Nucleases

In recent years, targeting modification of specific gene is becoming one of the powerful tools to elucidate gene function, generate disease models and transgenic livestocks. As a replacement of conventional gene targeting technology, Zinc finger nucleases (ZFNs) gradually attracted the attention of researchers. ZFNs made up of two domains, one is DNA binding domain composed with zinc finger proteins, the other one is cleavage domain composed with Fok I endonucleases. The ZFNs recognize target DNA by peptide-DNA affinity and fused Fok I nucleases generate double strand breaks (DSB), subsequently non-homologous end joining (NHEJ) results in small indels and leads to targeted gene mutation. In this study, we successfully knocked out mouse MSTN gene by engineered Zinc finger nucleases and obtained the MSTN gene knockout homozygous mice.1. To optimize the double plasmids transfection conditions of mouse embryonic stem cells (mESCs), Plasmids pDsRed2-1 and pIRES2-EGFP were digested by EcoRI and CpoI. The target fragments were ligated together to constitute a new red fluorescent protein (RFP) expression plasmid pRFP-N1. This plasmid combined with pEGFP-C1 which encodes the green fluorescent protein (GFP), were used to transfect the mESCs R1 mediated by lipofectamine LTX. To obtain the optimal transfection parameters, different DNA concentrations and liposome volumes combination were tested.36 hours after transfection, the cells that co-expressing green and red fluorescent protein can be observed in most of parameters. The result indicated that using lipofectamine LTX can achieve an efficient transfection of two plasmids for mES cells. The flow cytometry analysis showed that adding 500ng each of the plasmids,4 μL Lipofectamine LTX and 2×105 cells in 12-well cell culture plate can achieve the best co-transfection efficiency 26.9%.2. A pair of ZFNs was designed to recognize the target site that located at exon 1 of mouse MSTN gene. Each ZFN recognized the 18 bp DNA sequence, in between is a 6 bp cleavage site.Fok I endonucleases are designed as heterodimer variants, in order to provide an additional level of specificity to ZFN cleavage by effectively eliminating unwanted homodimerization events elsewhere in the genome. mESCs R1 were transfected with ZFNs using the best transfection parameters. Totally 768 single cells were picked, and 148 of them grew into colonies. The MSTN monoallelic mutations were identified in 8 colonies, and the biallelic mutations were identified in one of them. The knockout efficiency was reached 6.08%. The monoallelic mutation cell colony 1-3 was selected for second round of ZFNs transfection, and 2 cell colonies (D02, D08) that containing biallelic mutations were identified. The cell line 1-3 and D02 exhibited alkaline phosphatase activity, and were positively labelled by antibodies against Oct-3/4、Sox-2 and SSEA-1. SSEA-4 was negative in these two cell lines. These results indicated that these two cell lines maintained the undifferentiated mouse embryonic stem cell characteristics. Chimeric mice were generated from 1-3 and D02 cells by blastocyst injection. The sequencing results of the PCR products across the mutation sites confirmed that the mice containing the same mutationa as the injected cells.3. 1-2pL of ZFNs mRNA (20 ng/μL each) produced by in vitro transcription were microinjected into the cytoplasm of mouse pronuclear-stage embryos.28 pups were born and four of them (M4, M7, M8 and M24) were MSTN mutants. Three genotypes were identified in the cleavage site of the M8 mouse, which include the wild type and two mutation types, That means M8 mouse is a Chimera. The offsprings carrying a homozygous mutation were derived from M4 and M7, and two homozygous mutation offsprings from M8. However, M24 was infertile.The statistical results of birth rate indicated that MSTN homozygous mutation would reduce the pup number of birth. The body weights of homozygous mutation mice obviously higher than that wild type at 10 weeks of age. Meanwhile, there was a dramatic increase in skeletal muscle mass which is evident after the removal of the skin from homozygous mutation mice. The result of western blot showed that MSTN was almost undetectable in two homozygous mutation mice derived from M8. The results of real-time PCR showed that the mRNA level of the gene relevant to muscle development changed in different degree between homozygous mutation mice and wild type.