Study On Genetically Modified Cloned Tibet-minipig

Background and objectiveSince the anatomical and physiological similarites to humans and the relative ease with which they can be handled with in experiment, the minipigs are considered improtant model animals in biomedical research. The Tibet-minipig in this study coming from the farming and farming&herding region in attitude 2500-4300m of Qing-zang plateau, is the only one that can accommodate the high elevation climate. The closed geographical environment makes it keep the genuine breed. The Tibet-minipigs were introduced from Tibet to Guangzhou for laboratory animal research in 2004. Until now, we have finished acclimatization and experimental animalization. We also carried out lots of studies about animal model, drug experiment and transgenic cloning. The research results about immunology and genetics showed that this minipig presented unique characteristics and was an excellent experimental miniature pig.Preparation and research about genetic engineering animal is the one of key technologis in life science field. We can obtain different genetically modified animals by use of transgenic and gene knockout technology. Now the establishment of genetically modified mice with pronuclear microinjection and ES methods is the most mature and common technology. However porcine ES cells are not availabe in many labs, yet successful isolation has been reported recently. Pronuclear microinjection is also not suitable for pig because of hard to get fertilized eggs and excess fat drop. Therefore, it is difficult to achieve genetically modified pigs with the traditional methods.Since the advent of nuclear transfer technology, we can produce genetically modified pigs from gene modified somatic cells. In order to establish the research platform of gene modified Tibet minipig, here we report producing transgenic and gene knockout Tibet minipig.I. Establishment of porcine Oct-4 promoter-driven EGFP reporter system for evaluation of Oct-4 status in porcine pluripotenct cellsOct-4, a member of Class V of the POU (Pit-Oct-Unc) transcription factor family, plays a crucial role in establishment and maintenace of transcriptional regulation during early embryo development and cell differentiation. The Oct-4 protein contains a POU domain that binds to the octamer sequence ATGCAAAT located in the promoter or enhancer regions of its target genes, to regulate the expression of target genes.Oct-4 is a maternally inherited transcription factor and can be detected in matured oocyte. Oct-4 gene begins to be expressed after the genome activate. Until embryo implantation, its expression may stop gradually and only restricted in germ cells but not in differentiated somatic cells. Thus Oct-4 is a gatekeeper in regulateing the pluripotency of ES cells and in the beginnings of mammalian development.Then, how can monitor the in vivo Oct-4 expression? The common methods used include RT-PCR, westent blot, immunofluorescence assay and so on. But all the methods have the defective of cell death after treatment and hinder further research. In this study we utilized the method of Oct4-GFP report system, GFP driven by the porcine Oct-4 promoter. The endogenous Oct-4 expressing status can be detected and monitored by GFP fluorescence observation. This visualized method has been applied in research of mouse and human pluripotent stem cells, but not in pig.II. Establishment of TYR knockout albinism Tibet minipig by nuclear transferPig is usually used for research about skin burn, skin toxic experiment and allergic reaction of cosmetics and drugs because of the skin anatomical and physiological similarites to human. However, the skin color of most Chinese minipig is black or mixed colour, leading to unsuitable for research observation. Therefore the production of albinism Tibet minipig is key question for experimental animalization.The skin and hair color of mammalian depend on the type and content of pigment. Tyrosinase is the rate-limiting enzyme in the process of the pigment synthesis.TYR deletion and mutation would result in albinism in human, mouse, cat and so on. This study intend to produce TYR knockout albinism Tibet minipig with combination of somatic cell gene knockout and nuclear transfer. The albinism Tibet minipig will act as standard animal model for toxicity and anaphylactic response of cosmetics and drugs. Compared with traditional breeding method, it is more effective by use of breeding with transgenic technology. This method not only saves the breeding time but also produces strain with uniform genetic backgrounds.Methodsâ… . Establishment of porcine Oct-4 promoter-driven EGFP reporter system for evaluation of Oct-4 status in porcine pluripotency cells1.Isolation and identification of porcine Oct-4 promoter regionFor cloning porcine Oct-4 promoter, two primers were designed, namely oct-pro-F2/oct-pro-R2..Porcine Oct4 promoter DNA amplified was subcloned to pMD18T vector to create pT-Oct-pro for sequencing. DNA sequecne was identified by DNA blast.In order to further analyze the structure of porcine Oct-4 promoter, we made multi-alignment between the corresponding region of human, bovine, rabbit, mouse, rat. The functional domain of porcine Oct-4 promoter region can be predicted and analyzed according to the alignment results.2. Construction of pOGN2 vector and identification by transient transfection in vitroThe porcine Oct-4 promoter was isolated by digestion of pT-Oct-pro with Hindâ…¢and Kpnâ… and inserted upstream of EGFP cDNA in pEGFP-N2 (BD Biosciences Clontech) to produce pCOGN2. Then CMV promoter in pCOGN2 was removed by disgestiong with Aseâ… and Bglâ…¡. The DNA fragment with 3'-and 5'-protruding ends produced by Aseâ… and Bglâ…¡digestion was converted to that with blunt ends and was self-ligated with DNA blunting kit from Takara to create the vector pOGN2 for this research. The vector pOGN2 contained two expression cassette of Oct4-EGFP and Neo, which facilitate drug screening and fluoresence observation.Expression assay of porcine Oct-4 promoter in pOGN2 was carried out to identify the efficiency and specification of the promoter. Hindâ…¢lineared pOGN2 or lineared pEGFP-N2 were transfected into porcine IPS cells in which Oct-4 was expressed. After transfection, IPS cells and feeder cells were obsearved under fluorescence microscope.3.Establishment of Oct4-GFP transgenic PFF cell clonesPorcine fetal fibroblasts (PFFs) were isolated from 35d Tibet minipig fetus. G418 toxicity trails was performed before drug screecing. The concentration gradients were as follows: Oμg/ml,200μg/ml,400μg/ml,600μg/ml,800μg/ml,1000μg/ml,1200μg/ml,1400μg/ml. On the basis of cell death results, the optimized concentration was found out. For transfection, Hindâ…¢-lineared pOGN2 and lipo2000 (invitrogen) was mixed together. Then the transfection was performed according to the manufacturer's protocol. The day after transfection, cells were passaged to 100-mm dishes by the ratio of 1:25.At 2 days of culture, selective medium with 1000μg/ml G418 were added and cells were cultured for additional 7-10 days. G418 resistant cell colonies were selected by the cloning ring (sigma) and propagated in a 48-well culture plate.4-6 days later, wells in which cells growed confluently were trypsinized (60μl trypsin,0.5% EDTA) and seeded in a 24-well plate for frozen soon after. Parts of the cells were used for PCR screening. Cells were resuspended in lysis buffer and 1μl lysate was used in a 20μl PCR reaction. The positive cell clones were cryopreserved in liquid nitrogen for further use.4. Production of SCNT embryos with Oct4-GFP transgenic PFFs and establishment of Oct4-GFP transgenic pigIn vitro muturation of porcine oocytes:Immature oocytes are derived from ovaries obtained from the slaughterhouse and subsequently matured in vitro. Porcine ovaries are collected from an abattoir and transported to the laboratory in a thermos filled with saline maintained at 30-35℃within 4 h after collection. Follicular fluid from 3-6-mm antral follicles is aspirated by using an 18-gauge needle attached to a 10-mL disposable syringe. The oocytes are matured for 42-44 h at 39℃,5% CO2 in air. Oocytes with an intact plasma membrane, round shape and visible first polar body are selected and kept in manipulation medium until use.Nuclear transfer:Enucleation was accomplished by aspirating the first polar body and the metaphase II plate in a small amount of surrounding cytoplasm. Then injection of donor cells into the perivitelline space of enucleated oocytes was carried out. Electrical fusion and activation were achieved by direct current of 1.2kV/cm,60μs. After activation, embryos are transferred to 500 mL embryo culture medium in a four-well dish and covered with light mineral oil.In vitro culture of embryos and fluorescence observation:Oct4-GFP transgenic SCNT embryos were observed under fluorescence microscope after 5-7 days culture. Oct-4 protein was detected in GFP positive embryos with immunofluorescence.Embryo transfer:Since the nuclear transfer embryos are generally of a low quality, a large number(>100) one-cell stage embryos cultured 18-22 h with good shape (intact membrane) are surgically transferred into an oviduct of the surrogate.5.Porcine iPS cells induction with pOGN2 transgenic PFFsRetroviral vectors encoding EGFP or human Oct-4, Sox2, Klf4 and c-Myc were produced using HEK293T cells.4×104 PFF cells per p6 well-dishes were infected with 2.0ml each of unconcentrated retroviral vector in the presence of 8 mg/ml Polybrene. Two days after infection, the cells were trypsinized, and 1×104 cells were transferred onto feeders in a 10cm dish in dFBS medium. Emerging ES-like colonies were picked from 17-22 d and observed under fluorescence microscope. The GFP positive IPS clones were passaged for identification and further research.â…¡. Establishment of TYR knockout albinism Tibet minipig by nuclear transfer1.Construction of TYR gene knockout vector(1)Cloning of unknown TYR genomic DNA. According to the DNA multi-alignment among human, bovine and dog, two primers pairs were designed for each homologous arm. Nested PCR was performed to clone TYR gene as two homologous recombination (HR) arms. (2) Construction of TYR PNS gene knockout vector. Two homologous arms were disigned and cloned into the vector pSSC-9 that contains Neor and two HSV-tk genes as positve and negative screening genes, respectively. The knockout vector was designated as pSSC-tyrko.(3) Design and synthesis of TYR ZFN and identification in vitro, construction of donor plasmid. With the reference of mouse TYR ZFN sequence and the results of OPEN-ZiFit screening, one pair of ZFN specific for TYR was designed. Recognition DNA were synthesized with overlap PCR. The recognition sequence were ligated to different types of endonuclease (DD/RR, KK/EL).After constructing mutant EGFP vector encoding TYR ZFN binding sites, then we transfected the vector into 293T cells to generate 293T cell lines with an integarted mutant EGFP. For ZFN-mediated gene targeting experiments, TYR ZFN vector and GFP donor template plasmid were cotransfected into transgenic 293T cell line. With effective gene targeting and HR, the cell became GFP positive, as detected by flow cytometry.GFP positive cells were examined and compared between DD/RR,KK/EL by microscopy of confirm GFP expression and FCAS.2.Disruption of TYR gene in PFFs(1)Positive and negative TYR gene knockout strategyFor DNA transfection, appromate 1×107 cells were electroporated with 30μg Sfi I-linearized gene targeting vector DNA at 450v and 350 uf using a gene pulser II electroporator (Bio-Rad). Transfected cells were then split into twelve 100mm plates and were selected with 1000μg/ml G418 (geneticin, Invitrogen) 48h after transfection,2μM gancyclovir (GANC) 96h after transfection. Selection was carried out for-12 days. Then G418-GANC resistant colonies were selected by the cloning ring (sigma) and transferred to 48-well culture plates for expansion and analysis(2) ZFN mediated TYR gene knockout strategyTYR ZFN and TYR donor template plasmid were cotransfected into PFFs with lipo2000. Transfected cells were then split into 100mm plates by ratio of 1:25 and were selected with 1000μg/ml G418 (geneticin, Invitrogen) 48h after transfection,2μM gancyclovir (GANC) 96h after transfection. Selection was carried out for 12-15 days. Then G418-GANC resistant colonies were selected by the cloning ring (sigma) and transferred to 48-well culture plates for expansion and analysis.3.Production of TYR gene knockout pig by nuclear transferResultsâ… . Establishment of porcine Oct-4 promoter-driven EGFP reporter system for evaluation of Oct-4 status in porcine pluripotency cells1.Here we succeed in isolating 3.2kb DNA spanning 5'-flanking Oct-4 gene promoter region of Tibet minipig. The sequence of the promoter region is 97% homology to NCBI data (NW₀01886435). By alignment with Oct-4 promoter of human, bovine, rabbit, mouse, rat, four highly conserved promoter regions (CR1-4)were identified within the porcine region. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. The core promoter and distant enhancer located in CR-1 and CR-4 respectively, showing highly conserved at different domain. Proximal enhancer PE-1B located within CR2 and PE-1A outside CR2. PE-1A from different organism differed greatly,indicating that PE-1 related regulation is species specific.2. Vector pOGN2 was constructed successfully by identification with enzyme digestion and DNA sequencing. After transfection into porcine IPS cells, GFP only can be observed in IPS cell but not in feeder cell, while both IPS and feeder cell expressed GFP in contrast group transfected with pEGFP-N2. The transfection results showed that the pOGN2 merely expressed in Oct-4 active cells.3.PFFs were transfected with lineared pOGN2 and screened with G418. After identification with PCR,10 positive cell clones were achieved and 8 of them growed well.4. SCNT was carried out with the 8 pOGN2 transgenic PFFs. After observed at days 5-7, embryo with clone #3,#4,#8 expressed high GFP fluorescence, embryo with clone#1,#2 showed weak GFP, while no GFP fluorescence were detected in other clones. Oct-4 protein also can be detected in GFP positive embryos. Another SCNT was performed with cell clone#3,#4,#8.The SCNT embryos were transferred into an oviduct of the surrogate.5. To further verify Oct4-EGFP reporter system for monitering the endogenous Oct-4 gene, we initially transfected pig fetal fibroblasts with Oct4-EGFP and then reprogrammed Oct4-EGFP transgenic PFFs into iPS cell colonies by infection with retroviral vectors encoding the four factors (Oct-4, Sox2, Klf4, c-Myc). EGFP expression iPS-like cell colonies were detected by day 20. And many signle cells also expressed GFP but not formed colony. Nevertheless most colonies were EGFP-negative.â…¡Establishment of TYR knockout albinism Tibet minipig by nuclear transfer1.PNS mediated gene knockout(1)After two rounds of PCR, the unknown TYR DNA was cloned successfully. Sequence blast and analysis proved the cloned DNA was correct.(2) The targeted vector was constructed from 6kb TYR genomic DNA fragment spanned part of intron 1 (4.5kb) as a long homology arm and part of intron 2 as a short homology arm. PNS gene knockout vecotr pSSC-tyrKO was achieved successfully.(3) We obtained 166 clones surviving from G418 and GANC screening. A large portion of these clones were transgenic clones though the addition of GANC as negative screening drug. Only one clone of these was considered as TYR gene knockout clones after PCR test.(4) 400 SCNT embryos were produced with the gene knockout cell clone and transferred into 2 receptor gilt. One was pregnant and the other returned to estrus. When performing laparotomy at 36 days of pregency, several implantation sites were found and yet no fetus discovered for absorption. 2. ZFN mediated TYR gene knockout(1)We designed and engineered one pair of 3-finger ZFN tyrZFN-131F/tyrZFN-131R that targeted specific porcine TYR gene by overlap PCR method. pST1374-DD/RR and pST1374-KK/EL was constructed successfully.(2) Gene targeting and recombination was initiated by co-transfection of the expression plasmids for the pair of ZFNs and the EGFP donor template plasmid.3 days after transfection, the cells beame GFP positive,which was detected under fluorescence microscope.The number of EGFP positive cell in cells transfected with tyrDD/RR (0.23%) appeared to be lower than transfected with tyrKK/EL (0.29%). No GFP positive cells were observed post-transfection with GFP donor template plasmid only.ConclusionsI. Establishment of porcine Oct-4 promoter-driven EGFP reporter system for evaluation of Oct-4 status in porcine pluripotency cells1.cloning of 5'upstream promoter region of Tibet minipig Oct-4 gene2. Successful construction of pOGN2 containing Oct4-EGFP and NEO expression cassette and identification by IPS transfection.3. Establishment of pOGN2 transgenic PFFs cell lines4. SCNT embryos with pOGN2 transgenic PFFs cells expressed GFP, indicating that we have developed Oct4-GFP monitoring system. This system can be applied in porcine pluripotent research such as IPS and ES.II. Establishment of TYR knockout albinism Tibet minipig by nuclear transfer1.Cloning unknown TYR genomic DNA and construction of TYR PNS gene knockout vector. One gene knokcout cell clone was achieved by this method.2.Designing and engineering one pair of ZFN targeted TYR gene. This ZFN mediated gene correction of the mutant EGFP locus in 293T cells correctly. 3.Designing the mutant EGFP transgenic 293T cell model to access the efficiency of ZFN.Innovation points1.This study intent to develop gene modified cloning Tibet minipig with Chinese autonomous intellectual property since the Tibet minipig was the unique minipig breed of our country.2. It was the first time to monitor porcine endogenous Oct-4 expression with Oct4-EGFP system, in which the GFP was driven by the whole 5'upstream regulation region.3.It was first time to establish albinism Tibet minipig by nuclear transfer technology. This albinism animal model provied perfect big animal model for human albinism disease research.