| Background and objectiveDue to its small size, easy to grab a fixed, which facilities the experimental operation, and the similarity with human in the anatomical structure and physiological characteristics, minipigs has been widely used in biomedical research。Also its eating habits are very similar with humans as well part of the human food chain, so that its application in biomedical research with fewer ethical issues。In view of these advantages, it has become an important animal model for biomedical research. Tibet-minipig is one of the smaller mini-pigs in the world, which comes from the Qinghai-Tibet Plateau with altitude of2500-4300m, lived in the agricultural areas and semi-rural and pastoral areas, their ability to adapt to high altitude climate and grazing-based features, so it has a good resistance and a strong ability to adapt severed condition, in the same time, the closure of the geographical environment of Tibet mini-pig maintained its very pure genetic characteristics. our group introduced the Tibet mini-pig in2004from Tibet to Guangzhou, and has completed the acclimatization and experimental animal research, and carried out the study of animal models, drug experiments and transgenic research. From the study of immunology, genetics.we found that this strain has a unique immune markers and genetic features, coupled with its unique smaller size, which makes it an excellent experiment mini-pigs.Preparation of genetically engineered animals, using the technologies of molecular biology and transgene or knockout to change the genetic composition of animal genomes, is one of the forefront of key technologies in life sciences, which can modify the genetic traits of the animal from gene level, and then change the external phenotype of the animal. With the advancement in molecular biology techniques and the aging in cloned transgenic nuclear transfer technology, the dream to acquired engineered animals according to the requirement of people to be realized。 However, the current production of genetically engineered animals, confined to several kinds of animals。And the most mature technology is the study of genetically engineered mice, in which micro injection in situ and ES cells are commonly used techniques. Although ES cells of the pigs have been cultured and separated successfully, however, the culture system is not stable and far from reaching the level of promotion and application. The fertilized eggs of pig have limited sources contained too much fat, which hindered the micro-manipulation. Therefore, using these traditional methods is difficult to obtain genetically modified pigs. gene targeting is the technologies of integrating the foreign gene into the genome of target cells on certain sites at a fixed-point, thus changing genetic traits of the certain cells based on the principle of homologous recombination, Which Is an important molecular biological techniques developed in the1980s. The technology has the advantages of strong site-specificity and the stable genetic passage after the targeting, which is a powerful technical tools in the field of life sciences, genomics and disease researches。With the emergence of somatic cell nuclear transfer technology and gradually matured, transgenic technology has gradually become the main method for the preparation of transgenic and gene knockout pigs, Which applying molecular biological techniques and gene transduction skills to achieve the purpose of transgenic and gene knock, changing the genes composition in cultured somatic cell in vitro, and then using the SCNT to acquired gene-modified pigs。At present, the gene targeting strategy, including the positive and negative screening (PNS), the zinc finger nuclease-mediated (ZFN)gene targeting and adeno-associated virus-mediated(AAV) gene targeting. The most important factor affecting the targeting efficiency is the methods of transferring exogenous gene into cells。The traditional methods of gene delivery, such as:microinjection, liposome, electroporation, which have a very low probability of homologous recombination in eukaryotic cells, however, it has reported that adeno-associated virus-mediated(AAV) gene transfer strategy can overcome this shortcoming, and achieve a higher targeting efficiency. Recent studies have shown that the adeno-associated virus is a single-stranded small DNA virus, has the following advantages:①Non-pathogenic, host-free and relatively safe;②The ability to infect cells in a broad spectrum, including the split and non-dividing cells;③it has no specific integration, but less likely to occur insertional mutagenesis as well。④exogenous gene can have a stable expression, lasting up to2years.Since the skin of pig is very similar with that of human in anatomical structure and physiological function, Pig’s skin is often used in the researches of skin toxicity test, skin burns and allergic skin reactions of cosmetics and drugs. However, the skin or hair color of the most Chinese mini-pigs is black or mixed color, obviously affecting research observation. White pigs are more common in domestic pigs and other large pigs, which is not conducive to the experimental operation. Tibet mini-pig, due to its body size, the similarity with human in skin anatomy and physiology characteristics and its unique biological properties, is an excellent experimental mini-pig. However, Its skin and hair color is black, so it is in urgent need to developed its black skin and hair into a white ones to achieve the goal of cultivating the Tibet mini-pigs as an excellent Laboratory Animal.The skin and hair color of mammalian depends on the type and content of pigment. Tyrosinase(TYR) is the rate-limiting enzyme in the process of pigment synthesis, studies confirm that deletion or mutation of the tyrosinase gene will directly lead to albinism of the human, mouse, cat and other animals.Therefore, in this study, we intended to apply the adeno-associated virus-mediated gene targeting to acquire the albino Tibet mini-pigs with TYR gene knockout, based on the advantages of adeno-associated virus in gene transferring. Compared with the traditional genetic breeding methods, Transgenic breeding techniques, which can not only greatly shorten the breeding time, also obtain the animals with a single genetic background and a clear genetic characteristics, is a more effective method in cultivating the albino Tibet mini-pigs.Methods1. The identification and cryopreservation of isolated and cultured Porcine embryonic fibroblast (PEF) cells of Tibet mini-pigsEmbryos were obtained under sterile surgical procedure from the30-35d of pregnant Tibet mini-pig. The head, limbs and internal organs of the embryos were removed in clean bench. Embryonic tissue were cut into lmm3pieces using ophthalmic scissors. Trypsin was added to the tube to digest the tissue block, after it has been fully digested, adding DMEM containing10%fetal bovine serum (FBS) to terminate the digestion, then centrifuged and removed supernatant. After adding DMEM medium containing10%fetal bovine serum to resuspend cells, put the cells into culture incubator. The cells were digested till they grow to80%confluence, centrifuged, removed the supernatant and then added1.5-2ml of freezing medium, gradient cooling the cells and finally placed in liquid nitrogen for cryopreservation.2. Construction of the TYR gene targeting vector(1) Cloning of the targeting homology arm sequences of the TYR gene. According to TYR gene sequence from the previous study of our team, we the design for the exon-2of the TYR gene as knockout sites。The upstream750bp of exon2is homologous short arm and the downstream1500bp is long arm.The long and short arms were amplified by PCR.(2)Construction of TYR gene targeting adeno-associated virus (AAV) vector.Based on obtained TYR gene sequences, the long and the short homologous arms were cloned into the PMD-18T vector and were named Larm and Sarm respectively, and then identification by restriction endonuclease digesting and sequencing. (3)The amplification of the GFP-neomycin CDS,its promoter PGK and polyA: amplify the drug resistance gene and the green fluorescent protein gene(GFP),its promoter and poly A tail by PCR with the plasmid PGKneotpAlox2as a template, and primers were designed. Then the PCR products were identified with1%Agarose gel electrophoresis and the purposed DNA fragments were recycled; and then targeted DNA fragments were cloned into the PMD-18T vector (named as the PGK-GFP-neo-PA).(4)The amplification of fusion gene:plasmid Larm and Sarm as a template, the long and short homologous arms were amplified by PCR, the PCR products recycled (named larm and sarm). Amplify the fusion gene:Amplified the fusion gene with the three PCR products of larm, sarm and PGK-GFP-neo-PA as templates. The PCR products (named fusion gene) to be cloned into PMD-18T simple vector.(5)The ligation of fusion gene and the targeting vector:Fusion gene plasmid, which were sequenced correctly, was digested by the NotI restriction enzyme. The targeted DNA fragments were recycled (named S-neo-L). Plasmid pAAV-MCS was also digested by NotI restriction enzyme, the targeted DNA fragments were recycled (named AV-ITR).The fragment of AV-ITR was dephosphorylated, and then ligated with the S-neo-L fragment with T4DNA ligase, the sequenced correct ligation products were named AV2-TYR-KO.(6)The study of the transfection efficiency of different serotypes of adeno-associated virus (adeno-associated virus, AAV) in Tibet mini-pigs embryonic fibroblasts. Observing the transfection efficiency of different serotypes of adeno-associated virus infecting PEFs to obtain the serotype virus, which has the highest transfection efficiency to PEFs. and using this virus serotype to package virus will obtain the highest targeting efficiency.(7) packaging of adeno-associated virus(AAV).The helper plasmid AAV2-PDG and core plasmid were co-transfected into293T cells, and7days later to harvest the virus.(8)The PEFs were infected with the harvested adeno-associated virus and then observed the infection efficiency. 3. Screening and identification of positive knockout cells, after AV2-TYR-KO virus infecting PEFs.The adeno-associated virus were added into cell dishes after the PEFs were cultured2-3days at the MOI was105.After48hours of infection, the expression of green fluorescence protein w;ere observed, then screening positive clones with G418.The positive cells were subcultured and cryopreserved. Portion of positive cells were identified by PCR with the lysates as a template.4.TYR gene knockout Tibet mini-pig (TYR+/-) was obtained by by Somatic cell nuclear transfer technology (SCNT) with TYR gene knockout cells as nuclear donors.Results1.obtained the adeno-associated virus serotype of AAV2which has a high infection efficiency to PEFs.2.Successfully constructed TYR gene knockout adeno-associated virus vector AV2-TYR-KO.3. Successful packaging the TYR gene knockout adeno-associated virus AV2-Tyr-KO4.The initial attempt to applying virus AV2-TYR-KO to infect PEFs demonstrated a high infection efficiency.Innovation points1.In this project we use our unique Tibet mini-pig combined with somatic gene modification and cloning technology to construct genetically modified mini-pigs.2.Using adeno-associated virus as a gene transfer carrier has a higher targeting efficiency than traditional methods.3.According to the Tyrosinase (TYR) gene structure and functional characteristics, preparing gene modified albino Tibet mini-pig by somatic cell nuclear transfer technology and gene targeting, which can provide standardized large experimental animals for the research of Cosmetics, medicines, skin toxicity experiments and allergic reactions and also for human albinism pathogenesis and gene therapy research. |
