Detection And Analysis Of Fat1 Gene Targeting Cashmere Goat

Fat1 gene encodes omega-3 polyunsaturated acid dehydrogenase,which could transform polyunsaturated fatty acids from omega-6 to omega-3 to improve the content of omega-3 PUFAs in animals.But omega-3 polyunsaturated fatty acids could not synthesized by human body because of the enzyme deficiency.Therefore,Fat1 is widely applied to breed improvement the meat quality of the domestic animals.MSTN is a member of the transforming growth factor superfamily.It is a negative regulator which is associated with muscle growth and development.The MSTN gene mutated animals showed muscle hypertrophy which is called "double muscle phenomenon".Double muscle animals are growing faster,higher lean meat rate and lower fat content than ordinary animals.CRISPR/Cas9 has been widely used in gene knockout,gene knockin and multi point knockout simultaneously because of its low cost,simple operation and high cutting efficiency.In 2016,a gene targeting Cashmere goat which Fat1 gene integrated into MSTN gene by using CRISPR/Cas9 technology was born.PCR,Southern Blot,Western Blot,Real-Time quantitative PCR,fatty acid content,muscle tissue section,blood routine and blood biochemical analysis were used to detect the gene targetingd goat.The result showed that Fat1 gene was integrated into MSTN gene.1)The detection of exogenous gene integrationThe blood of Fat1 gene targeted Cashmere goat was collected to extract genome for PCR.And the ear fibroblasts of the Fat1 targeting Cashmere goat were isolated for the Southern Blot.The results showed that the exogenous gene Fat1 was inserted into the genome successfully and the Fat1 gene was integrated into the MSTN locus.2)The analysis of the Fat1 gene and MSTN gene expressionThe Real-Time Quantitative PCR was used to detect the relative expression of Fat1,MSTN,muscle-related gene and fat-related gene in the ear fibroblasts,muscle and fat tissues of the gene targeting Cashmere goat.The results showed that the relative expression of MSTN gene was lower than the control group.The muscle-related genes Myo D,Myf5,Myf6 and MEF2 C were higher than the control group.Fat1 gene was higher than the control group.The fat-related genes ACC,FASN,Fabp4,SREBP1,PPAR gamma and SCD were lower than the control group.3)The analysis of WeightThe birth weight,weaning weight and villus weight of the gene targeting Cashmere goats were measured respectively.Compared the homologous Cashmere goat with the same age male wild type control group,the weight of the gene targeting Cashmere goat was slightly higher than the control.4)The analysis of fatty acidThe fatty acids of Fat1 of Cashmere goats were identified by gas chromatography,including the omega-6 polyunsaturated fatty acids,omega-3 polyunsaturated fatty acids and the proportion of omega-6/omega-3.The results showed that the omega-6 polyunsaturated fatty acids of Fat1 gene targeting Cashmere goat were 0.63 times of the control;the n-3 polyunsaturated fatty acids were 1.10 times as high as the control group;the n-6/n-3 polyunsaturated fatty acids were 0.57 times of the control group.5)The analysis of the diameter of muscle fiberThe Fat1 gene targeting and the control Cashmere goats were analyzed by paraffin section.The result showed that the diameter of the Fat1 gene targeting Cashmere goat was larger than the control Cashmere goat.6)The analysis of blood routine and blood biochemistryThe blood routine and blood biochemical analysis of Fat1 targeted Cashmere goats and control Cashmere goats showed that there were no significant differences in blood physiology,liver function,renal function,blood glucose,blood lipid and myocardial function.The results showed that the exogenous gene Fat1 was successfully integrated into the MSTN gene locus.The expression of Fat1 and MSTN gene changed the expression pattern of the animal fat-related genes and muscle-related genes,which affected the body weight and fatty acid content of the animals.And the genetic changes did not affect the health of the gene targeting Cashmere goat and the study provided a new idea for the livestock breeding.