Generation Transgenic Minipigs With B Lymphocyte Specific Expression Of C-Myc Gene By Lentivirus-mediated Gene Transfer

Research background and significance1.cancer is a major disease killer of humanbeings.In twenty-first Century,cancer is almost become the first killer of human beings,has attracted great attention of people all over the world.Nowadays,the cancer death rate of urban and rural residents in China has been a high level of the world,and showed a sustained growth trend.The tumor has become the main cause of death of residents in china.China’s government and WHO has listed tumor as one of the key issues urgent need to solve.2.The occurrence and development of tumors is a slow process of evolutionAs everyone knows,carcinogenesis is a multifactorial process and different stages have different gene alterations.Generally speaking,the growth stage of tumor,can be divided into two parts:subclinical and clinical.Subclinical period is relatively hidden,contains tumor induction time.Different tumors undergo differentinduction times:someundergo several months,such as lymphoma,someundergo years,such as lung cancer,colon cancer and breast cancer etc.According to the statistics,calculating from the start time,the majority of human tumors in theperiod of sub clinical are 8-20 years,also can be 30-40 years.In addition to the clinical stage,it can be say that the occurrence,development and metastasis of tumor is a slow process of evolution.So we can use the animal model of tumor to study the various aspects of tumor.The role and limitations of human tumor model of Genetically engineered mice(GEM)At present,there has been a large number of GEM strains carrying human oncogene or anti oncogene.There has been mouse models of cancer genetics and pathology teaching resources database,the GEM model made great contribution to study the pathogenesis of tumor.However,mouse tumor models have some limitations.First of all,mice are different with human beings in anatomy,physiology,biochemistry and metabolism,the information obtained from mice can not fully reveal the true incidence of human tumor;secondly,because mice’s life is short,mouse tumor models can not fully simulate the whole dynamic process of tumor ’s development,metastasis and recurrence;thirdly,in the Biopharmaceutical Research and Development Process,there have been a lot of experimental data were confirmed that the safety evaluation of new drugs on mice is not very reliable.The agencies all around the world have Already prescribed that research and development of new drugs must do safety and toxicity evaluation and prediction at least in one nonrodent big animal before entering clinical trials.The large experimental animals usually are monkeys,dogs and pigs.4.Creation of Genetically engineered minipigs tumor modelThrough the comparison of the three kinds of commonly used large-scale experimental animals(minipigs,Beagle dogs and monkeys),we can find that mini pigs have obvious advantages in terms of price,ethical restrictions,mature time,pregnancy,litter size and strategy and technology of genetic modification.Our country is very rich in miniature pig resources and there are a number of minipigs breeds.So we choose minipig as our research object and Create Genetically engineered minipigs based on them.In view of the actual situation of our country,our research group planed to build a lymphoma model based on the minipigs.Because the results of the mice showed that compared to other solid tumors,lymphoma is more easily induced,and the time consuming is relatively short.Non Hodgkin’s lymphoma(NHL)accounts for the majority of lymphoma.In our country the proportion is about 90%.B cell type lymphoma accounts for the majority of clinical NHL and the proportion is about 70%～85%.So our group planed to establish lymphoma transgenic pigs based on B lymphocytes.C-myc gene was first discovered in Burkitt’s lymphoma patients;c-myc is a very strong oncogene and over-expression in many tumors;c-myc is thought to regulate the expression of all 15%genes.In addition,studies have confirmed that over expression of c-Myc in specific organs of transgenic mice can induce tumor occurrence in the corresponding organs,such as lymphoma,liver cancer and breast cancer,etc.And our group had previously proved that single over expression of c-Myc gene can make MiniPig embryonic fibroblasts undergo malignant transformation.in addition,Some scientists have established lymphoma mice and zebrafish model based on the expression of c-Myc transgenic mice and zebrafish.5.present situation of transgenic minipigs Mediated with Somatic Cell Nuclear Transfer method or Lentivirus-based Vectors methodAt present,there are two main methods to Produce transgenic pigs:somatic cell nuclear transplantation and method Mediated with Lentivirus vector.Nowadays,transgenic somatic cell nuclear transfer method has become the most mature method to create transgenic pigs,which is widely used.The letter method need to inject concentrated lentivirus into the subzonal of zygotes.After microsurgical operation training,common technicians can grasp the injection skills.With the advantages of easy to operate and higher transgenic efficiency,this method,as long as soluting the problem of how to get embryos at one cell stage,can be widely used.To sum up,our group originally intended application of somatic cell nuclear transfer method to establish B lymphocyte specific expression of c-myc transgenic pigs.But in the production process,we encountered some insurmountable problems.Finally we selected using lentiviral vector method to build B lymphocyte specific expression of c-myc transgenic pigs.We hope lymphoma will formation in transgenic miniature pigs,and to create lymphoma minipig model based on them.MethodPart one Application of somatic cell nuclear transfer method to establish B lymphocyte specific overexpression of c-Myc transgenic pig1.Construction of a lentiviral vector pEμ-c-Myc-IRES-mRFP(pEMIR)Taking pLV-c-Myc-IRES-EGFP plasmid as a template,PCR amplification of c-Myc gene,cloned into the pEμ-VH-miR155plasmid and amplification,obtained pEVC.Taking pEVC plasmid as a template,PCR amplification of Eμ-VH-c-Myc gene,cloned into pHIV-H2BmRFP plasmid and amplification,obtained pEVCHR.Taking pEVCHR plasmid as a template,PCR amplification of Eμ-VH-c-Myc-H2BmRFP gene,cloned into the pCDH-EF1α-MCS-GFP-Puro plasmid and amplification.Finally we obtained the target plasmid pEμ-c-Myc-IRES-mRFP(pEMIR).2.verification functions of the vector in vitroAfter verification,transformation,amplification,extraction and purification of,pEMIR were respectively transiently transfected into 293T,OCL-LY3 and OCL-LY10 cells.Inverted fluorescence microscope was used to detect the expression of green and red fluorescence.when it came to appropriate time we collected cells and extracted total RNA and protein.qRT-PCR and Western blot were used to detect c-myc expression and to verify the function of pEMIR in vitro.3.minipig embryonic fibroblast cells(PEFs)with EMIR transgene were preparedSwiss packaging system was used to produce lentivirus.when it came to 72 hours,the supernatant was collected and centrifugated at a low speed and filtrated.Lentivirus infected Tibet minipig PEFS and Bama minipig PEFS.Puro was used for selecting GFP positive cells,as the nuclear transfer cells for mononuclear.Part two Application of Lentivirus vector method to establish B lymphocyte specific overexpression of c-Myc transgenic pig1.Construction of a lentiviral vector harboring c-Myc gene under the control of Eμ-VH promoterTaking pCDH-μ-VH-c-Myc-H2BmRFP-PGK-GFP-Puro plasmid(pEMIR,CTC0119-1)as a template,PCR amplification of Eμ-VH-c-Myc gene,cloned into the pGFP-PGK-Puro(CTC0192-2)plasmid and amplification.The positive colony were selected and recombinant plasmids were extracted.Then we took advantage of restricted enzyme to digest the plasmid and sequenced it.Finally we obtained the target plasmid pEμ-VH-c-Myc-GFP-PGK-Puro(pEVMGPP).2.verification functions of the vector in vitroAfter verification,transformation,amplification,extraction and purification of,pEVMGPP were respectively transiently transfected into 293T,OCL-LY3 and OCL-LY10 cells.Inverted fluorescence microscope was used to detect the expression of green fluorescence.when it came to appropriate time we collected cells and extracted total RNA and protein.qRT-PCR and Western blot were used to detect c-myc expression and to verify the function of pEVMGPP in vitro.3.Production,concentration and titer determination of lentivirusUsing a conventional Swiss packaging system to produce lentivirus.pEVMGPP,packaging plasmid and shuttle plasmid were co-transfected into 293T cells.The supernatant was collected,centrifugated at a Low speed and filted when it came to 72 hours.Lentivirus Concentrated by Ultracentrifugation precipitation method.The viral titer was determined by fluorescence counting method of genechem company,but two methods are used in counting the number of fluorescent cells.One method is to use the inverted fluorescence microscope,the other is to use flow cytometry to detect fluorescence ratio of cells,and counte total cells.4.establish transgenic mice with B lymphocyte specific over expression of c-Myc and identificationIn order to ensure that concentrated virus titer is high enough to create transgenic minipig under the existing experimental conditions,we first use the concentrated virus in mice to obtain transgenic mice.Super ovulation female mice bred to male mice and collectin fertilized eggs.Concentrated lentivirus injected into subzonal of fertilized zygotes.Then the fertilized eggs were implanted into the oviducts or uterus of pseudopregnant femalemice to let them develop until delivery.After obtaining the filial generation mice,we detected GFP expression in blood cells by the inverted fluorescence microscope and flow cytometry and apply PCR to identify whether the transgenic c-myc gene was successfully integrated into the mouse genome to obtain the c-myc transgenic mice,that is c-myc transgenic founder mice.(1)inverted fluorescence microscope and flow cytometry was used to screen and identify the GFP positive blood cells in the peripheral blood of mice:Peripheral blood(PB)was collected at the eye socket of mice after 3 weeks of birth.The blood was used to detect the potential GFP positive cells by inverted fluorescence microscope and flow cytometry after splitting the red blood cells,removing the red blood cells and washing.Using the results to judge whether GFP was integrated into the mouse genome,to realize the preliminary screening of c-Myc transgenic mice.(2)apply PCR to detecte the integration of c-Myc gene in mouse genomePB genomic DNA of potential c-myc transgenic mice and wild-type mice(negative control)were extracted,taking them and plasmid pEVMGPP as a template respectively to PCR amplified gene fragments of GFP and Puro to identify genotypes of the potential c-myc transgenic mice.5.establish transgenic minipigs with B lymphocyte specific over expression of c-Myc and identification5.1 Establishment of transgenic pigs with B lymphocyte specific expression of c-Myc by the method of lentiviral vector and identification of the transgenic pigsSuper ovulation female Tibet minipigs bred to boars and collectin fertilized eggs.Concentrated lentivirus injected into subzonal of fertilized zygotes.Then the fertilized eggs were implanted into the oviducts or uterus of pseudopregnant female Tibet minipigs to let them develop until delivery.After obtaining the filial generation minipigs,we detected GFP expression in blood cells by inverted fluorescence microscope and the flow cytometry and apply PCR to identify whether the transgenic c-myc gene was successfully integrated into the minipigs’genome to obtain the c-myc transgenic minipigs,that is c-myc transgenic founder minipigs.(1)inverted fluorescence microscope and Flow cytometry was used to screen and identify the GFP positive blood cells in the peripheral blood of minipigs:Peripheral blood(PB)was collected from the ears margin intravenous of minipigs after 3 weeks of birth.The blood was used to detect the potential GFP positive cells by inverted fluorescence microscope and flow cytometry after splitting the red blood cells,removing the red blood cells and washing.Using the results to judge whether GFP was integrated into the minipigs’ genome,to realize the preliminary screening of c-Myc transgenic minipigs.(2)Apply PCR to detecte the integration of c-Myc gene in minipigs’ genome:PB genomic DNA of potential c-myc transgenic minipigs and wild-type minipigs(negative control)were extracted,taking them and plasmid pEVMGPP as a template respectively to PCR amplified gene fragments of GFP and c-Myc to identify genotypes of the potential c-myc transgenic minipigs.5.2 Establishment of transgenic pigs with B lymphocyte specific expression of c-Myc by B lymphocytes reinfusions after infected with pEVMGPP and identification of transgenic pigsPeripheral blood(PB)was collected from the ear vein of 3 weeks or more minipig.B lymphocytes separated by the flow cytometry sorting labeled pig CD20 and fluorescent antibody.Cultured B lymphocytes and join the IL-4 and CD40Lto activate lymphocytes.LV-EVMGPP infect B lymphocytes and reinfusion back to the minipigs.The blood of the minipigs regularly extracted to detect the survival of the infected B lymphocytes by using inverted fluorescence microscope and flow cytometry.6.Breed c-Myc transgenic minipigs and detection Genetic and expression stability of c-Mycc-Myc transgenic founder minipigs mated with wild type minipigs to obtain F1 minipigs.Inverted fluorescence microscope and flow cytometry was used to screen and identify the GFP positive blood cells in the peripheral blood of F1 minipigs to judge the expression stability of c-Myc.Apply PCR to detecte the integration of c-Myc gene in F1 minipigs ’ genome to determine whether c-Myc can Stable Transmission indirectly.Results:Part one Application of somatic cell nuclear transfer method to establish B lymphocyte specific overexpression of c-Myc transgenic pig1.Enzyme digestion and sequencing identification proved pEMIR was successfully generated.2.293T,OCL-LY3 and OCL-LY10 cells transiently transfected with pEMIR confirmed that the promoter Eμ-VH can regulate the expression of c-Myc and mRPP,PGK can regulate the expression of GFP and Puro,which confirmed the function of pEMIR in vitro.3.Acquired the GFP Tibet minipig PEFs and Bama minipig PEFs,which can be used as nuclear donor cells for nuclear transplantation.Part two Application of Lentivirus vector method to establish B lymphocyte specific overexpression of c-Myc transgenic pig1.Enzyme digestion and sequencing identification proved pEVMGPPP was successfully generated.2.293T,OCL-LY3 and OCL-LY10 cells transiently transfected with pEVMGPP confirmed that the promoter Eμ-VH can regulate the expression of c-Myc and GFP,which confirmed the function of pEVMGPP in vitro.3.Production,concentration and titer determination of lentivirusLV-EVMGPP infected human diffuse Large B Cell Lymphoma Cell Line OCL-LY3.green fluorescence could be observed under inverted fluorescence microscope at 48h,indicating that the virus has been successfully produced,which also showed that the transgene c-Myc can express.Before the virus was concentrated,the titer was 105 TU/ml with the number of fluorescence cells was counted under inverted fluorescence microscope;the titer was 5.32 x 104 with the number of fluorescence cells was counted with the method of flow cytometry.After the virus was concentrated,the titer was 7.193x107 TU/ml with the number of fluorescence cells was counted with the method of flow cytometry.And the titer was close to 108 TU/mlIwhich could be used to prepare transgenic minipigs.4.establish transgenic mice with B lymphocyte specific expression of c-Myc and identificationTwo hundred and eighty-eight fertilized eggs were selected and injected with lentivirus in Subzonal.Two hundred and seventy embryos survived and transfered into nine pseudopregnant mice,in which six mice were pregnant.Total of eighteen mice were obtained.the number were:3317-3332.The expression of GFP in the blood cells of 5 mice were found by Inverted fluorescence microscope and flow cytometry,the number of which were 3317,3319,3322,3325 and 3328.PCR and sequencing test confirmed that exogenous genes c-Myc and GFP were integrated into the genome of the five mice.In all,a total of five founder mice were obtained.5.establish transgenic minipigs with B lymphocyte specific over expression of c-Myc and identification5.2 Establishment of transgenic pigs with B lymphocyte specific expression of c-Myc by B lymphocytes reinfusions after infected with pEVMGPP and identification of transgenic pigsLV-EVMGPP successfully infected B lymphocytes of Tibet minipig and reinfusion back to the minipigs.