| BACKGROUND&OBJECTIVEIn 2004,Professor Weiwang Gu(Laboratory Animal Center,Southern Medical University)introduced Tibetan pigs to Guangzhou for acclimatization;it took 5 years to breed Tibetan pigs into a new experimental miniature pigs,named Tibet minipigs.Because of small size of body,easy to operate,and anatomical,physiological and biochemical similarites to humans,at present minipigs are becoming the most popular used large laboratory animals.At the same time,The reproductive performance,physiological and biochemical parameters and genetic polymorphism of the mini pig in Tibet were studied comprehensively and systematically.Tibet mini pig is China first one of the smaller size of the miniature pig,source in the Qinghai Tibet Plateau,elevation 2500-4300 meters in rural and agriculture and pastoral areas is only can adapt to high altitude climate and grazing of pigs and the closed geographical environment make Tibet mini pigs have the germplasm resources of non Chang Chunzheng.Due to small size,easy to operate and anatomy,characters of physiological and biochemical characteristics and people very close,miniature pig has gradually become biomedical research field of experimental animals,the number of applications by the substantial increase.At present,the development of transgenic animals is crucial in the field of life science technology,through gene knockout and transgenic technology change the composition of animal genes,can in the designer's idea to change the genes in animal experiments,meet the designers of the experiment the experimental conditions of genetically engineered animals.At present,transgenic and knockout studies were the most mature in mice,and the common methods were the nuclear microscopic injection and ES cells..And in pigs,embryonic stem cells,although there are some successful cases,but due to the pig embryo cell culture unstable,and did not get the level of industrialization and large-scale production promotion.In addition,the source of the pig's fertilized eggs is still relatively limited,and there are too many fat droplets in the pig fertilized eggs,but also very unfavorable for the original nuclear micro injection..Thus,it is difficult to obtain the gene knockout and transgenic pig by using the traditional methods of the prokaryotic microscopic injection and ES cells..With the continuous maturation of somatic cell nuclear transplantation,more and more transgenic cloning pigs and the pig genetically knock out are prepared by the technology..The method is mainly by genetic engineering methods of somatic cell nucleus gene modification of knockout,and then using the technology of somatic cell nuclear transfer of porcine body horizontal gene modified modifier genes cloned pigs.In somatic cell modification time(transgenic pig donor nucleus transplantation gene vector and for the stream of research progress),somatic cell types are many:at present,commonly used in transgenic pig vegetation of the nuclear donor cell types are:source in the cells of an individual's reproductive system,such as cumulus cells and granulosa cells,fallopian tube epithelial cells,primordial germ cells,immature Sertoli cells.Although these cells can provide nuclear,but in the method is still difficult to obtain,or by surgery,etc.,the operation time is longer,the difficulty of the work.The cells of the non reproductive system derived from the adult or the newborn individual,such as skin fibroblasts,porcine ear fibroblasts,muscle cells,tail tip fibroblasts and fat cells,etc..Here cells in methodology,obtain relatively simple,as long as by disinfection and local anesthesia,you can get the cells and the cells obtained from adult or newborn pigs,and fetal into fiber cells,no need to wait for more than 30 days and delivered from the em'bryonic,and surgical operation is easy to get,ready access to cells.But these cells also exist some disadvantages,in general we somatic cell nuclear transfer donor cells,we need the in vitro generation at least 10 generations above,as nuclear transgenic donor cells can carry a series of functional verification.And to knock out the animals,then need to more algebra in the body.Therefore,although the class of cells is simple,but need to be a nuclear donor is still need to carry out the validation of the body and the outside generation.The cells derived from porcine embryos,such as porcine fetal fibroblasts.The characteristics of this kind of cell mainly,value-added ability is exuberant,passage number,karyotype stability,easy to isolation and culture,and the formation of reconstructed embryos developmental ability is relatively strong,cultured for a long time.Such cells are also more likely to get,but also need surgery,and each obtained are to kill a pregnant sows cost and obtain cell sex need later were identified.(4)from the cells of the lymphatic system,such as terminally differentiated B cells,T cells;these cells with abundant sources,materials and convenient to transport and easy in vitro amplification,and specific binding in vivo specific antigenic points,and many other advantages.The stem cells,such as bone marrow stem cells,embryonic stem cells(ESCs es),induced pluripotent stem cells(induced pluripotent stem cells(IPS).This type of cells has the ability of unlimited proliferation,self-renewal and multi differentiation..Jin(Ref.31)think,in pigs,compared porcine fetal fibroblast cells,bone marrow mesenchymal stem cells the nucleus donor to obtain higher blastocyst rate and better quality of embryo,but stem cell line establishment,storage is a very difficult technology,the technical and financial requirements are very high,so this makes stem cells in transgenic and gene knock in technology use is very limited.In the selection and donor cells that typically need to be considered objective,animal welfare,drawing is convenient,in the process of producing transgenic pigs,especially gene knockout animal models,we usually choose to in vitro culture exuberant,screening for a long period of time,relative more easily obtained from fetuses fibroblasts.Cre/lox P system with real tight on/off ffunction of the control system,which is composed of a CRE and cre specific recognition of LOX P sites;when cells exist at the same time CRE and two LOX P sites in the same direction,LOX P sites between DNA fragments were excised,and then through the distal two sticky ends connected by recombinant DNA[3,4,40-42].Strategies(Figure 3)(illustrations attached to the reference):the transcription termination signal box(transcription stop cassette,at both ends of each of a LOX P sites Cre/lox P system mediated by conditional gene expression and in the same direction)is placed in the start between transgenic and objective,(the nucleus)no CRE protein,the downstream target genes of the stop cassette the expression and when(nucleus)CRE exists,mediated by recombinant DNA removed stop signal box,and then induced dormancy transgene expression.Liver expression of multiple proteins are associated with animals of many physiological and pathological phenomena,for the expression of the protein of conditional knockout is one of the related research.The Cre-LoxP recombinant enzyme system provides a powerful means that we can study specific gene function in specific tissues and organs as well as study the model of various diseases..And at present,the transgenic rats of Cre recombinant enzyme,which are specifically expressed by various tissues,are established..But the size of rats and human beings in the size of the individual or in the physiological and biochemical convenience are all the larger,while the pig and the rat is more close to the human.The liver specific expression of albumin promoter(albumin promoter,CRE expression was established by the method of in-fusion cloning vector and eventually established and turn the liver specific expression of Cre recombinase swine fibroblast cell line,for the next step to establish a Cre recombinase transgenic pigs lay the foundation.And the different promoter driven Cre recombinase can reach the regulation of gene expression in time and space,and showed the specificity of tissues and cells,but the start of specificity in the end how strong and Cre recombinase to mediated recombination and recombination efficiency is much less,there is no systematic evaluation criteria.Regarding this,this article through the establish the vector pCALL2-IRES-mCHERRY genetically modified(GM)in Tibet/Bama miniature pig,dedicated to the integration efficiency of evaluation of tissue specific CRE transgenic pigs.Based on the unit holding Tibetan cultivars/Bama miniature pig fetal into fiber cell separation passage to verify whether to several passages in vitro for production of transgenic and knockout with the exception of the modification.The isolation and culture of porcine ear fibroblasts which can replace the fetal fibroblasts for the nuclear test were verified..(a)Alb-Cre transgenic mini pigs and the evaluation of Cre activity of Z/RFP transgenic mini pigsSerum albumin is a protein synthesized in the liver of mammalian cells,and it accounts for about 5%?10%of the new proteins in the liver..Serum albumin can only be synthesized by the differentiation of mature hepatocytes,while the other tissues of the albumin gene in the shutdown state(the mouse albumin promoter tissue specific functional studies).The specificity of this tissue specificity of the albumin was studied by many scholars..The study found that albumin synthesis by liver growth and development of,in the development process of synthesis with the passage of time and liver development,albumin concentration increased gradually,to adulthood reached the highest.The expression of albumin was regulated at the transcriptional level..In view of the tissue-specific promoter in exogenous gene and tissue specific expression and gene therapy have important potential,this article by albumin start in porcine embryos into fiber cells within the functional activity of verification,for the future establishment of a liver specific expression of transgenic cloned pigs lay the foundation.So how to detect the exogenous Alb promoter has not been transferred to the cell interior.There is no activity of the porcine fetal fibroblasts on the albumin promoter,and how to monitor whether the cell carrier is the donor nucleus for the nuclear transfer of the donor cells?Green protein Puromycin(PURO)and fluorescent PGK(GFP)were used to regulate the expression of and the T2A was used to regulate the expression of the promoter and the..Among them,the resistance gene PURO can be used to screen out the positive cells,and GFP can be used to detect whether the screening of the drugs are positive.(two)the establishment of Tibet miniature pig ear fibroblast cell linesThe technology of somatic cell nuclear transfer has great potential for development of agriculture,biology basic research,biopharmaceutical and human medical research..There are a variety of animal cloning,wherein,nucleus donor cell is one of the key factors affecting the efficiency of nuclear transplantation.While the current nuclear donor cells with porcine fetal fibroblasts(porcine fetal fibroblast cells,PFFs),obtain the into fiber cells need will be pregnant 35 days of sows after cesarean section from embryos.After estrus,mating after 35 days of pregnancy to take a long time to wait,have an impact on the pace of the experiment.In addition,the cells were repeatedly frozen and the proliferation of subculture was affected..The research is focused on the isolation of fibroblasts from the ear tissues of newborn pigs,as the nuclear donor cells,and to obtain the stable cell line by experiments..And the test of the ability of the body is being carried out.Method(1)Alb-Cre transgenic porcine embryonic fibroblasts and its functional verification1 vector pAlb-Cre-PGK-GFP-T2A-PURO(pACGP)and pCALL2-IRES-RFP(pCIR)Construction of 1.1 vector pACGPUsing plasmid pAlb-Cre-GH/BS as the template,the PCR fragment of Alb-Cre was amplified and the gel was recycled,and the In-Fusion was cloned from Nhe to/CIa I pCDH-EF1-MCS-GFP-Puro..1.2 recognition of vector pCIRUsing the enzyme BGL II and ECOR V plasmid pCIR and Xho I,ECORI were identified,identification of correct again on the key components of the location of primers were designed.After sequencing,sequencing results with the expected sequence alignment.2 functional verification of pCIR and pACGPThe recognition of the conformity of the 2.1 vector pCIRWith plasmid pCIR transfected 293T cells and porcine embryos fibroblast cell PEF(porcine embryonic fibroblasts),48 to 72 hours in an inverted fluorescence microscope observation of fluorescence,backward X-gal(beta-galactosidase staining staining.The functional verification of the recognition of the 2.2 vector pCIR and Cre recombinant enzymeCells and 293T were transfected with pLV-CIG and PEFs by vector pCIR and,and red fluorescence,green fluorescence and X-gal were observed for 72 hours..3 functional verification of pCIR and pACGP3.1 pACGP constructs the liver specific expression Cre porcine fetal fibroblast cell linePackaging plasmid PMD2G and PsPAX2 and pACGP to 8/3:5:20/3 proportion(unit:UG)Co infected 293T cells,60 hours after collection virus supernatant,centrifuged and filtered virus supernatant was used to infect PEFS,72 h after the infection was observed by fluorescent,followed by puromycin selection,until all the cells with GFP expression,select stop.3.2 vector pCIR constructs LoxP expression of transgenic porcine fetal fibroblasts lineBy electroporation method of carrier pCIR transfection PEFS.After transfection,24 hours of 1:20 passage,after passage with G418 screening for 2 weeks,screening concentration is 1mg/ml,maintain concentration for 500ug/ml and maintained for I week.The selected cells were stained with X-gal,and the identification of the cells was successful..(two)the establishment of Tibet miniature pig ear fibroblast cell linesTake new Tibet mini pigs born 1-2 weeks,nail clipping cover size of pig skin,after penicillin and streptomycin mixture by adding 5%(penicillin Streptomycin Solution PS)phosphate buffer flushing(phosphate buffered saline(PBS)washing 3 times,then with collagen enzyme digestion overnight,centrifugation to remove collagenase,after vaccination in 25-cm2 culture flasks of subculture and cell morphology observation.Result:(1)Alb-Cre transgenic porcine embryonic fibroblasts and its functional verification1 vector pAlb-Cre-PGK-GFP-T2A-PURO(pACGP)and pCALL2-IRES-RFP(pCIR)Construction of 1.1 vector pACGPThe construction of vector pACGP was successful,and was confirmed by sequencing and enzyme digestion..1.2 recognition of vector pCIRThe vector pCIR was confirmed by sequencing and enzyme digestion.2 functional verification of pCIR and pACGPThe recognition of the conformity of the 2.1 vector pCIRThe expression of PEF and green fluorescence of 293T cells and porcine embryonic fibroblasts were observed by 48?72 and pCIR hours after fluorescence inverted microscope..After X-gal staining,some cells were stained blue.The functional verification of the recognition of the 2.2 vector pCIR and Cre recombinant enzymeCarrier pCIR and pLV-CIG cotransfection of 293T cells and PEFS,72 hours of infection,were observed under the inverted fluorescence microscope,found that some cells expressing red fluorescent protein,part of the cells expressed green fluorescent protein and underwent X-gal staining cells were stained blue.3 ACGP-PEFs and CIR-PEFs were established by pACGP and pCIR respectively.3.1 pACGP constructs the liver specific expression Cre porcine fetal fibroblast cell linePackaged lentivirus was visible 90%293T cells expressed green fluorescence and infection after PEFS cells in cells expressing green fluorescent,by puromycin screening all positive cells and by PCR to confirm the integration of target gene.3.2 construction of pCIR using vector CIR-PEFsThe cells were stained with X-gal after the electric switch,and the whole cells were stained blue.(two)the establishment of Tibet miniature pig ear fibroblast cell linesThe morphology of the cells was normal and the passage time was about 3 days.Conclusion:(1)Alb-Cre transgenic porcine embryonic fibroblasts and its functional verificationACGP-PEFs and CIR-PEFs has been established,and verified in line with expectations,as the donor somatic cell nuclear transfer to establish liver specific expression of cre of Tibet mini pigs and evaluation of CRE transgenic pigs Z/R of transgenic pigs.(two)the establishment of Tibet miniature pig ear fibroblast cell linesThe fibroblast cell line of the new pig ear was basically established and was further verified by the new generation.. |
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