Generation Of NLRP3 Genetically Modified Pigs And Establishment Of RYR2 Mutant HiPSC And Preliminary Differentiation Into Cardiac Muscle

(1)Generation of NLRP3 gene modified pigs using TALENs/CRISPR technologyBackground:Genetically modified disease-resistant pigs and high-quality pigs have enormous economic prospects and social benefits.Since pig embryonic stem cell lines have not yet been established,a common method to produce cloned pigs is to combine somatic genetic modification with somatic cell nuclear transfer technology.However,due to the limited expansion ability of somatic cells and the low efficiency of somatic cell gene targeting,the fixed-point genetically modified pigs is difficult to obtain.Fortunately,the development of gene editing technologies,such as ZFN,TALENs,and CRISPR,has made gene-based modifications more efficient.A genetic mutation(R262W)on 262th amino acid of the human NLRP3(NACHT,LRR and PYD domains-containing protein 3)gene causes activation of NLRP3 inflammatory bodies and produces an autoinflammatory disease.Mice,pigs and humans are conserved in the NLRP3 mutation site.In transgenic mice that mimic human R262W mutations,NLRP3 inflammatory body activation did not show a severe inflammatory response,but increased enhancement that leading to increased disease resistance.It is suggested that we can prepare cloned pigs with enhanced disease resistance by simulating the same mutation of the NLRP3 gene in pigs.In addition,we intend to simultaneously produce the NLRP3 knockout pigs,and compare the physiological indicators of NLRP3 knockout pigs with fixed-point mutant pigs,and build large animal models to study NLRP3 inflammasome-related disease mechanism.Method:We built TALENs and CRISPR gene modification vector separately and screen ones with high gene editing activity on porcine fibroblasts.Then we use the gene modification technology and homologous recombination technology to prepare NLRP3 gene-modified porcine fibroblast cell lines.Next,the somatic cell nuclear transfer technology(SCNT)is operated and the recombinant embryos are cultured in vitro and transplanted into recipient sows to produce of NLRP3 knockout or site-directed transgenic pigs.RESULTS:We constructed a TALENs targeting vector targeting the porcine NLRP3 gene,as well as single-stranded DNA(ssDNA)used as a template for homologous recombination,and screened for the most efficient TALENs combination.At the same time,a CRISPR gene editing system,including targeting vector for porcine NLRP3 gene and corresponding ssDNA,were constructed.Using the CRISPR/Cas9 gene modification technology,we obtained 10 NLRP3 knockout porcine fibroblast cell lines.Using the CRISPR/Cpf1 gene modification technology combined with homologous recombination technology,we obtained three porcine fibroblast cell lines with NLRP3 point-fixed mutagenesis.Conclusion:We mimic the human naturally occurring NLRP3 gene mutation,and combine TALENs/CRISPR gene modification technology with homologous recombination technology and somatic cell nuclear transfer technology,obtaining NLRP3 genetically modified pigs successfully.(2)Establishment and preliminary differentiation of RYR2 mutant iPSCsBackground:The emergence of induced pluripotent stem cells provides new ideas for the establishment of disease models.Since it can be induced from many of the patient's own readily available cell sources(eg,urine;dermal fibroblasts)and has similar pluripotency to embryonic stem cells,hiPSCs can be used to create personalized disease models without ethical issues,greatly expanding the scope of precision medicine.Catecholamine polymorphic ventricular tachycardia type I(CPVT1)is a hereditary heart disease caused by a single gene mutation in the ryanodine receptor type 2 gene(RYR2).Mutations in the RYR2 gene cause a disturbance in cardiac function,characterized by stress-induced ventricular arrhythmias,which lead to a large number of young people to die each year.Therefore,the establishment of iPSCs of patients with hereditary heart disease caused by RYR2 single gene mutation and differentiation them into cardiomyocytes is of great significance for the study of disease mechanisms and drug screening.Methods:We use a lentiviral system to integrate Yamanaka s four factors(Oct4^Sox2,Klf4,and Nanog)into skin fibroblasts from patients with catecholamine polymorphic ventricular tachycardia type I(CPVT1)and induced them into iPSCs.Then verify the hiPSCs pluripotency by morphology,surface antigen,gene expression,and epigenetic status of pluripotent cell-specific genes.Then,iPSCs were differentiated into cardiomyocytes in vitro,and the expression of specific markers of cardiomyocytes was identified,thereby establishing a genetic heart disease model caused by RYR2 single gene mutation.Results:We identified the genotypic characteristics of a catecholamine polymorphic ventricular tachycardia type I(CPVT1)patient,and induced two hiPSCs from the patient's skin fibroblasts.By staining stem cell markers,it can be seen that hiPSCs exhibit strong alkaline phosphatase activity and express Oct4 and Nanog as well as embryonic stem cell-specific surface antigens,including stage-specific embryonic antigen-3(SSEA3),stage-specific embryonic antigen-4(SSEA4),tumor associated antigen(TRA-1-60)and tumor associated antigen(TRA-1-81).By analyzing the demethylation degree of Oct4 gene's promoter region of hiPSCs,it was confirmed that the promoter regions of both two hiPSCs,iPS-2 and iPS-4,were largely demethylated.The expression of pluripotency genes in hiPSCs was significantly higher than that in fibroblasts by real-time fluorescent quantitative PCR(P<0.01).The expression levels of three germ layer marker genes of embryoid bodies(EB)differentiated from hiPSCs were 5 to 50 times that of hiPSCs by RT-PCR,which proved that hiPSCs we obtained have the potential to differentiate into three germ layers in vitro.HE staining of teratoma sections obtained by inoculation of non-obese diabetic/severe combined immunodeficiency(NOD-SCID)mice with hiPSCs demonstrated that hiPSCs have the potential to differentiate into three germ layers in vivo.We also determined that iPS cells were a single cell source by tandem repeat technique(STR)detection,and there was no cross-contamination.The above proves that the hiPSCs we induced are highly pluripotent.Then we differentiated iPSCs into cardiomyocytes,and spontaneous cell pulsation clusters were observed after 11 days of differentiation.Using RT-PCR,we found that the expression of early genes related to myocardial development in beating cell clusters was 20-40 times higher than that of hiPSCs.Conclusion:We successfully induced two specific iPS cell lines of catecholamine polymorphic ventricular tachycardia type I(CPVT1)and demonstrated that they have strong pluripotency.We initially differentiated iPS cells into cardiomyocytes,and observed myocardial cells with rhythmic contraction and high expression of myocardial specific markers.However,due to the preliminary exploration stage of this study,the myocardial differentiation program needs to be improved,and the functional verification experiment has not been involved,and further research is needed.