Vascular Smooth Muscle Cell Specific Scap Gene Knockout Causes Embryo Experimental Study Of Abnormal Cardiac Outflow Tract Development

Objective:Sterol regulatory element binding protein lysis activator（SCAP）,also known as cholesterol sensitive organ,plays an important role in cholesterol regulation in the body.Previous studies mainly focused on lipid metabolism.In recent years,the understanding of this gene in development has been further deepened,especially the abnormal changes in various organs caused by embryo.In this study,the effect and mechanism of this gene deletion in the cardiac outflow tract of transgenic mouse embryos were discussed.Methods:In this study,transgenic mice with SCAP knockout using Cre/loxp system to construct vascular smooth muscle cell specific expression of Cre enzyme（SM22-Cre）were selected as tool mice,including SCAPflox/flox and SM22-Cre^+/-mice.Female SCAP^flox/floxlox/flox was mated with male SM22-cre^+/-mice,and SM22-cre was obtained.SCAP^flox/+,that is,vascular smooth muscle specific SCAP knockout mice required for the experiment.DNA identification was performed to screen the required genotypes and verify the knockout effect.The obtained SM22-cre/SCAP^flox/+transgenic mice were mated,and the embryonic mouse tails were identified at E9.5,E10.5,E11.5,E12.5,E13.5,E14.5d,E16.5d,and E18.5d,and the proportions of homozygous,heterozygous,and wild-type mice were counted.The embryonic development of homozygousmiceandwild-typemicewasobservedunder stereomicroscope.E10.5d homozygous and wild-type mouse outflow tracts were then collected for transcriptome sequencing to detect the influence of gene expression and signaling pathways related to heart development.Results:1.In studies on embryonic development abnormalities caused by SCAP knockout,all identified offspring mice were heterozygous mice(SM22-cre/SCAP^flox/+)or other wild-type mice.No homozygous mice(SM22-cre/SCAP^flox/flox)were born,indicating that the homozygous mice had died in the embryonic stage.2.According to the gene identification results,before E14.5d,the ratio of homozygote:heterozygote:wild type was 1:2:1,and after E16.5d,the ratio of homozygote was 0.In the anatomy of outflow tract at various stages,the outflow tract and right ventricle were found at E9.5d and E10.5d,and the SCAP knockout group became shorter and narrower.3.Due to E10.5 outflow tract abnormality,the outflow tract and right ventricular tissue were taken for RT-PCR.Transcriptome sequencing results showed that 16,810 differentially expressed genes were found,including 12,693 up-regulated genes and 4,117 down-regulated genes.The analysis of the signal pathway showed that the differentially expressed genes related to embryonic heart development were up-regulated.Conclusion:Vascular smooth muscle cell-specific SCAP gene knockout affected the abnormal development of outflow tract and right ventricle of homozygous mice during the embryonic period,resulting in cardiac malformation,resulting in the birth rate of homozygous mice being 0.The exploration of its mechanism in transcriptome sequencing may be related to the abnormal signal pathway of embryonic heart development,lipid metabolism pathway,cell mitosis,inflammation and immunity caused by the gene deletion.