The C3 Knockout Pig Model And The Establishment Of A Human Embryonic Stem Cell Line Stably Expressing Red Fluorescent Protein Were Constructed Using CRISPR/Cas9 Technology

BackgroundThe complement system is the major effector of the branch of the immune system.Research on complement now includes more than 30 soluble and cell-bound proteins.The complement-reaction sequence starts with an enzyme cascade.Complement system takes part in specific and nonspecific immune mechanisms of the body,carrying out a number of basic functions:lysis of cells,bacteria and virus;opsonization,which promotes phagocytosis of particulate antigens;inflammation,fragmentation binding to specific receptors on cells of the immune system cause inflammation;immune clearance,which removes immune complexes and apoptotic cells.Three established pathways of complement activation exist,including the classical,alternative,and lectin pathways.C3 is the central component of the complement system and primarily synthesized by hepatocytes,in addition,other tissues and cells also have the capability to synthesize C3 such as macrophages,dendritic cells,polymorphonuclear leukocytes,proximal tubular epithelial cells,fibroblasts,uterine epithelia,pneumonocytes,and activated T cells,indicating that it may possess pleiotropic physiological function.Previous studies have reported that C3 is involved in some diseases,like glomerular disease,hemolytic uremic syndrome,gastric carcinoma,and rheumatoid arthritis.To date,C3 knockout rodent animal models like mouse and guinea pig have been reported.However,these animal models have limitations due to their physiology and gene expression,which differ from humans,and studies involving large C3-deficient animal models have not been conducted.Pigs have similarities with humans in terms of anatomy,physiology,immunology,and clinical relevance.Moreover,pigs are more ethically and economically acceptable than other animals.Recently,the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)system has significantly improved gene targeting efficiency and has been extensively used to generate genetically modified animal models in various species,including mice,rabbits,sheep and pigs.In the present study,we used the CRISPR/Cas9 system combined with the somatic cell nuclear transfer(SCNT)technology to generate C3 targeted piglets.ObjectiveTo generate C3 KO pig models with CRISPR/Cas9 system.Methods1.We find C3 gene of pigs from genbank,and designed two target sites based on CRISPR/Cas9 guideline to conduct C3-Cas9 plasmids.Compared the target efficiency of two plasmids,the higher efficiency plasmid was transfected into PFFs by using nucleofection program and screened monoclonal cells for PCR sequencing.2.C3 KO cell pools were used as donors transferred into oocyte by somatic cell nuclear transfer(SCNT)technology.Reconstructed embryos were transferred into the uterus of surrogate pigs to generate C3 KO piglets.3.PCR sequencing was used to identify piglets genotypes;Western Blot and ELISA were used to detect the content of C3 protein in serum;IHC was performed to analyze the expression of C3 protein in tissues;The complement hemolysis 50%assay(CH50)was performed by using a liposome immunoassay technique;Serum cytotoxicity was tested with human 293T cells by using WT pig serum,heat-inactivated WT pig serum and the serum from C3 KO piglets.Results1.According to C3 gene sequence from genbank,two pairs of C3-Cas9 plasmids were designed and constructed.Then transferred into early passage male primary PFFs and screened C3 biallelic mutations cell lines,two groups of surrogate pigs were transferred by SCNT.6 piglets were produced from the first group and 13 piglets were generated from the second group.Totally we got 19 cloned piglets.2.All of the piglets were detected as C3 bialletic mutations and the genotypes were all the same with donor cells.There was no C3 protein in cloned piglets serum detected by western blot and ELISA.Also there was no C3 expression in tissues of cloned piglets by IHC.The results showed that the CH50 of the C3 KO piglets was not detectable compared to that of the WT piglets,indicating there is no complement activity in C3 KO piglets serum.In addition,serum cytotoxicity was indicated that the cytotoxicity of C3 KO piglet serum is similar to the heat-inactivated WT serum,indicating there is no any complement activity in the C3 KO piglet serum.ConclusionUsing CRISPR/Cas9 system combined with SCNT technology,we generated C3 bilallelic mutation and complement deficient pigs.This is the first report for C3 KO pigs,which could be utilized as a valuable large animal model for the elucidation of the roles of C3.BackgroundHuman embryonic stem cells have the potential of renewal and differentiation,which can differentiate into any type of cells and tissues.In 1998,the establishment of human embryonic stem cells was an important revolution.Engineering approaches to confer human stem cells with novel genetic circuits that monitor and control specific cell behaviours may help to known more about human stem cells.However,difficult cultivating,hard to transfect,and some others restricted the research of genetics in hESCs.Nucleofector technology transfected DNA into nuclear which increased the efficiency of transfection.In the early year of 2005,the efficiency was reported as high as 20%-65%in human stem cells.Because of the promoter sensitivity,gene was easy to slience after transfection.Jennifer obtained the human embryonic stem cell(hESC)lines labeled with red fluorescent protein(RFP)used EF1a promoter and passaged 10 times.Stem cells are divided into naive pluripotency and primed pluripotency.Stem cells from inner cell mass of mouse were regarded as naive pluripotency,and from epiblast were considered as primed pluripotency.Human stem cells are similar to epiblast stem cells and thought as primed.Primed cells failed to chimaeras and could not to develop a whole body.Human stem cells will be used in clinical and be a useful tool of organ regeneration.We obtained the hESC lines labeled with RFP used Nucleofector technology and detected the pluripotent of RFP-hESCs.ObjectiveTo establish the human embryonic stem cell lines labeled with red fluorescent protein,and identified differentiation potential of the RFP-hESCs in vitro.Methods1 The pEFl a-DsRed-Express2 plasmid was transfected into hESCs by using nucleofection program and screened monoclonal cells with G418 drugs.2 Perform alkaline phosphatase staining,immunofluorescent staining and RT-PCR to detect the pluripotent of RFP-hESCs.3 Identify differentiation potential of the RFP-hESCs in vitro.4 Induce Primed cells to Naive cells.Results1 We have successfully obtained the hESC cell lines stably expressing RFP.The RFP-hESCs possessed pluripotent of stem cells.The expression of Oct4?Sox2?Klf4?Nanog were no obviously defference between hESCs and RFP-hESCs.2 The RFP-hESCs formed to embryonic body and possessed the potential of 3 germ layers differentiation.It also can form to definitive endoderm and qRT-PCR showed the pluripotent transcription factors were almost not expressed.3 We obtained Naive-like cells which could not passaged.Conclusion The hESC cell lines stably expressing RFP has been successfully established and can differentiate into other cell types.The cell lines can be used for subsequent research.