Using CRISPR/Cas9 To Generate Homozygous Bmp9 Knockout C57BL/6 Mice

Caused by chronic liver injury,hepatic fibrosis is common in alcoholic hepatitis,fatty liver and viral hepatitis,which may turned into necrosis and liver cirrhosis.Many factors associated with hepatic fibrosis have been reported to be regulated by BMP9,be it directly or through many pathways.Therefore,BMP9 can be developed as a potential biomarker.BMP9 is a king of growth differentiation factor secreted by hepatocyte.Previous research mainly focused on its roles in stem cell differentiation and osteogenesis process.The way how BMP9 worked in the process of chronic hepatic fibrosis is unclear.The third generation of gene editting technique CRISPR/Cas9 is very helpful for bioscience.Researchers can edit or modify any protein and gene of interest by merely designing sgRNAs.Knockout and knockin can be conducted easily.We generate homozygous BMP9 knockout C57BL/6 by microinjecting BMP9 sgRNA and Cas9 m RNA.Protocal has been optimized.Our work will contribute to further exploration of BMP9 and other transgenic model animals.Results show as below.1.Establishing CRISPR/Cas9 knockout model mice.We download information about BMP9 gene and its cds sequence from NCBI and Ensemble database.The online sgRNA design platform scores candidate sgRNAs.We will use the one which ranks the top.Then transcribing and purifying BMP9 sgRNA using PCR production.Microinjecting BMP9 sgRNA and Cas9 mRNA into zytotes of C57BL/6 mice.Transplanting the live ones into ICR receptors.2.Establishing homozygous BMP9 knockout mice.Genotyping the F0 mice after being delivered to pick out the frameshift mutants.Back cross breeding was conducted to generate homozygous knockout mice.Phenotyping,Western Blotting and immunohistochemical methods are combined to confirm it.Also,testing levels of BMP9 expression in F3 mice by Western Blotting is necessary.Above all,sgRNA targeting at the exon1 on BMP9 gene is designed,its activity tested.Mice generated are tested in three levels,phenotyping,genetyping,and protein expression.It is confirmed that transgenic mice we generated express a much lower level of BMP9.After testing the protein amount expressed by F3 mice,we can say that homozygous BMP9 knockout model C57BL/6 is established.