WO2018205641A1 - 一种抗寒及瘦肉型转基因猪及其制备方法 - Google Patents

一种抗寒及瘦肉型转基因猪及其制备方法 Download PDF

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WO2018205641A1
WO2018205641A1 PCT/CN2017/120153 CN2017120153W WO2018205641A1 WO 2018205641 A1 WO2018205641 A1 WO 2018205641A1 CN 2017120153 W CN2017120153 W CN 2017120153W WO 2018205641 A1 WO2018205641 A1 WO 2018205641A1
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transgenic
pigs
transgenic pig
cold
lean
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French (fr)
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赵建国
郑千涛
秦国嵩
姚婧
曹春伟
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中国科学院动物研究所
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Definitions

  • the invention relates to the field of genetic engineering, in particular to a cold-resistant and lean-type transgenic pig and a preparation method thereof.
  • breeding has gone through routine selection of quantitative genetics to molecular marker-assisted selection (MAS) and genotypic or quantitative trait locus (QTL) direct selection based on genomics and molecular biology.
  • MAS molecular marker-assisted selection
  • QTL quantitative trait locus
  • uncoupling protein 1 is a protein that is specifically expressed in brown fat and is present on the mitochondrial inner membrane. It can eliminate the formation of electrons in the inner layer of the mitochondria due to the respiratory chain. The difference in proton concentration allows this electrochemical concentration potential to be dissipated as heat, which is also referred to as non-shaking heat.
  • pigs do not contain brown fat (Jastroch, M., & Andersson, L. When pigs fly, UCP1makes heat. Molecular Metabolism, 4(5), 359-362. doi: 10.1016/j.molmet.2015.02.00
  • the present invention provides a cold-resistant and lean-type transgenic pig which is a transgenic pig overexpressing the UCP1 gene in adipose tissue.
  • the UCP1 gene may be selected from UCP1 genes functional in any species, such as human, bovine, sheep, mouse, and the like.
  • the UCP1 gene is an uncoupling protein 1 gene derived from a mouse, and is specifically activated by an adiponectin promoter.
  • the adiponectin promoter is a murine adiponectin promoter.
  • the UCP1 gene and promoter in the donor plasmid of the present invention are digested by a Pcdna3.1 vector comprising a murine adiponectin promoter and a UCP1 coding sequence, and the nucleotide sequences thereof are SEQ ID NO. 2 and SEQ ID NO, respectively. .3 is shown.
  • the present invention also provides a method for preparing the aforementioned transgenic pig, comprising the following steps:
  • the constructed donor plasmid and the Cas9/gRNA vector are co-transfected into the porcine fetal fibroblasts, the monoclonal cells are obtained by the limiting dilution method, and the positive cells successfully labeled are further identified by PCR;
  • the positive cloned cells obtained by the screening are used as nuclear donor donor cells, and the isolated oocytes are nuclear transfer recipient cells, and the somatic cell clones are obtained by nuclear transfer technology to obtain transgenic pigs.
  • the starting vector of the donor plasmid can be a vector conventionally used in the art.
  • the pLB vector is selected as the starting vector.
  • the target sequence in the donor plasmid is located before the promoter sequence and the UCP1 gene is located after the promoter sequence.
  • the target sequence was designed using the online design CRISPR Design Tool (http://crispr.mit.edu/) developed by Professor Zhang Feng of the Massachusetts Institute of Technology and submitted to Thermo Fisher for its nucleotide sequence as SEQ ID. As shown in NO.1, the exon 2 of the porcine uncoupling protein 1 pseudogene is located in the pig genome.
  • the Cas9/gRNA vector is prepared according to conventional techniques in the art.
  • the PX330 plasmid containing Cas9 was purchased from Addgene.
  • the PX330 plasmid was first cleaved with BbsI endonuclease, and the linearized plasmid was purified and recovered.
  • the gRNA recognition sequence ie, the aforementioned target sequence
  • the gRNA recognition sequence is subjected to denaturation and renaturation treatment to change from single-stranded nucleotide to double-stranded oligonucleotide, and then double-stranded DNA and linearized PX330 plasmid are expressed by T4 DNA ligase. Make a connection. Transformation, plating, shaking, further sequencing to identify positive bacterial liquid, large extract plasmid spare.
  • the present invention provides the use of the aforementioned method for increasing the cold resistance of a transgenic pig, and/or increasing the lean rate of the transgenic pig, and/or increasing the feed conversion rate of the transgenic pig.
  • the raw materials or reagents referred to in the present invention are all commercially available products, and the operations involved are routinely operated in the art unless otherwise specified.
  • the present invention obtains transgenic pigs which are resistant to cold stimulation and fat deposition and which have an increased lean rate by transferring the uncoupling protein 1 gene of the mouse into the genome of the pig.
  • the present invention provides a transgenic pig that is both resistant to cold and capable of increasing lean meat rate through fat deposition reduction.
  • the invention improves the two important production traits of pigs by manipulating the single gene, and not only lays a foundation for the application and basic research of large animal gene editing, but also brings new ideas for breeding workers to improve the traits of livestock.
  • Figure 1 shows the targeting vector construction and gene knock-in identification strategy of the present invention.
  • Figure 2 shows the identification of positive targeting cells of the present invention.
  • Figure 3 is a genotype identification of transgenic pigs of the present invention.
  • Figure 4 is a graph showing changes in the temperature of the anus of the pig under the frozen condition of the present invention.
  • Fig. 5 is a view showing the infrared photograph and body surface temperature of pigs under the frozen condition of the present invention.
  • Figure 6 shows the detection of slaughter indicators of wild type and transgenic pigs of the present invention.
  • the UCP1 expression vector Pcdna3.1 initiated by the murine adiponectin promoter was obtained by cloning the murine adiponectin promoter and the UCP1 coding sequence into the Pcdna3.1 plasmid backbone. Sequence determination and primer synthesis were performed by Thermo Fisher. Taq enzyme, T4 DNA ligase and endonuclease were purchased from Beijing NEB Co., Ltd., and the reagents for somatic cell nuclear transplantation were purchased from Sigma. Refer to "Molecular Cloning (Third Edition)" for routine experimental procedures such as digestion, ligation, recovery, transformation, and PCR amplification.
  • the gRNA recognition sequence was designed using the online design tool CRISPR Design Tool (http://crispr.mit.edu/) developed by Professor Zhang Feng of the Massachusetts Institute of Technology and submitted to Thermo Fisher, which is located in the porcine uncoupling protein. 1 exon 2 of the pseudogene.
  • the PX330 plasmid containing Cas9 was purchased from Addgene. The PX330 plasmid was first cleaved with BbsI endonuclease, and the linearized plasmid was purified and recovered.
  • the gRNA recognition sequence is then subjected to denaturation and renaturation treatment to change from a single-stranded nucleotide to a double-stranded oligonucleotide, and the double-stranded DNA is ligated to the linearized PX330 plasmid using T4 DNA ligase. Transformation, plating, shaking, further sequencing to identify positive bacterial liquid, large extract plasmid spare.
  • the Pcdna3.1 plasmid was double-digested with KpnI and XhoI endonuclease, and the adiponectin promoter-UCP1 element was recovered.
  • the adiponectin promoter-UCP1 fragment was ligated to the pLB vector using the pLB Zero Background Rapid Cloning Kit (Tiangen Biochemical Technology Co., Ltd.), and the positive plasmid was identified.
  • the obtained new plasmid was digested with BspEI enzyme, purified and recovered, and the gRNA recognition sequence carrying the BspEI cleavage site, ie, the target sequence (Bait sequence), was ligated to the linearized plasmid by T4 DNA ligase.
  • a donor plasmid was obtained.
  • Digestion composition DMEM high glucose medium (Gibco) added 15% fetal bovine serum (Hyclone), 0.032% collagenase IV (Sigma), 25Kunitz units/mL DNase I (Sigma) and 40 ⁇ g/mL gentamicin ( Sigma). After the digestion was completed, the digested product was collected, centrifuged at 3000 rpm for 10 min in a centrifuge tube, the supernatant was discarded, resuspended in the medium, and centrifuged.
  • Digestion composition DMEM high glucose medium (Gibco) added 15% fetal bovine serum (Hyclone), 0.032% collagenase IV (Sigma), 25Kunitz units/mL DNase I (Sigma) and 40 ⁇ g/mL gentamicin ( Sigma).
  • DMEM high glucose medium Gibco
  • Hyclone fetal bovine serum
  • 40 ⁇ g/mL gentamicin Sigma
  • the fetal fibroblasts were resuscitated two days before transfection into a 25 cm2 culture dish.
  • the cells were transfected until the cells grew to about 70%, and the medium was replaced 4 h before transfection.
  • the cells were digested with 0.25% trypsin (Invitrogen), resuspended in electroporation, and the Cas9/gRNA plasmid and the donor plasmid were added to the resuspended cells.
  • Transfection was carried out using a nuclear power transducer (Nucleofector 2b Device, Lonza) using the U-023 procedure.
  • the transfected cell suspension was transferred to a 60 mm culture dish and cultured for 48 hours.
  • the cells were digested with 0.25% trypsin (Invitrogen), the digestion reaction was terminated, and the cell density was measured using a hand-held automated cell counter (Millpore). Cells were seeded into 96-well plates using limiting dilution according to cell density. 100 ul of medium was added to the wells and only one cell was contained per 100 ul of medium. After 3-4 days, change the solution and transfer the cells to the 24-well plate when the cells are full in the 96-well plate. Part of the cells in the 24-well plate are transferred to the 6-well plate, and the other portion is further cultured in the 24-well plate. For genotypic identification. Finally, a total of 26 cell clones were obtained and identified by PCR. It was found that the transgenic positive clones were amplified at the 5' junction and the 3' junction, and the positive clones were clones 8, 14, and 17 (Fig. 2). For subsequent experiments.
  • the pig's ovaries were collected from the slaughterhouse and placed in a thermos flask containing physiological saline containing penicillin and streptomycin at 37 ° C and returned to the laboratory within 1 hour. After washing the ovary with physiological saline for 3-5 times, the follicles with a diameter of 3 to 8 mm on the ovary surface were taken, and the follicular fluid containing the oocytes was collected in a 50 mL centrifuge tube and washed with TL-HEPES egg washing solution for 3 times.
  • COCs cumulus-oocyte complexes
  • the oocytes matured in vitro for 42-44 hours were digested with 0.1% hyaluronidase (purchased from Sigma) to remove cumulus cells around the oocytes, and the cytoplasm was uniformly collected under a stereo microscope.
  • the oocytes of the first polar body that is, the MII stage oocytes, are obviously placed in the microscopic operation drops, and the mature oocytes are fixed with a fixed tube so that the polar body is located at 3 o'clock, and the outer diameter is 20 ⁇ m.
  • the denuclear needle aspirates the first polar body and about 1/8 of the cytoplasm (including the nucleus); injects the UCP1 transgenic fibroblast into the perivitelline space of the enucleated oocyte, so that the donor cell and the egg
  • the mother cell membrane is contacted; then the oocyte is fused with the donor cell using an electric activation method (BTX Electro-cell Manipulator 200) to obtain a reconstituted embryo.
  • the reconstituted embryos were placed in PZM3 solution, cultured at 39.0 ° C, 5% CO 2 for 14 to 16 hours, and then subjected to embryo transfer.
  • the reconstituted embryos cultured in vitro for 14-16 hours were transferred to the fallopian tubes of the pseudo-pregnant sows, and about 180 embryos were transplanted per sow. After 28 days, the sow was pregnant by ultrasound. If pregnant, ultrasound is monitored every two weeks for pregnancy. The invention obtains 3 pregnant sows and successfully produces 12 piglets.
  • GM pigs and wild-type pigs were placed in a 4° refrigerator and frozen for 4 hours.
  • the anus temperature of pigs was measured by an electronic thermometer (Tianjin Jinming Instrument Co., Ltd.) every 1 hour (Fig. 4). It can be seen from Fig. 4 that the cold temperature of the transgenic pigs was significantly higher than that of the wild type pigs between 1 h and 4 h.
  • the pigs were photographed with an infrared camera (FLIR) to record changes in the temperature of the pig's body surface (Fig. 5). It can be seen from Fig. 5 that after cold stimulation, the infrared display color of the transgenic pigs is significantly stronger than that of the wild type, that is, the temperature transgenic pigs in the white line marker region are higher than the wild type.
  • FLIR infrared camera
  • the slaughter experiment was carried out when the pigs were 6 months old. Fasting 24 hours before slaughter. Weighed before slaughter and sacrificed by anesthesia. Head, hooves, tail and internal organs (retain kidney and oil), weigh, and record as weight. The ratio of weight to pre-slaughter weight is the slaughter rate.
  • the pig carcass was separated from the middle, and the left carcass was separated, and the lean meat, fat, skin and bone were separated and weighed separately, and the proportion of each part in the total was calculated (see Fig. 6). It can be seen from Fig. 6 that there is no difference in the slaughter rate between wild-type and transgenic pigs, and the lean meat rate of the transgenic pigs is significantly higher than that of the wild type.
  • the invention provides a cold-resistant and lean-type transgenic pig and a preparation method thereof.
  • the present invention obtains a transgenic pig which is resistant to cold and can reduce lean meat rate by reducing fat deposition.
  • the transgenic pigs provided by the present invention are both resistant to cold and can reduce lean meat production by reducing fat deposition.
  • the invention improves the two important production traits of pigs by manipulating the single gene, and not only lays a foundation for the application and basic research of large animal gene editing, but also brings new ideas for breeding workers to improve the traits of livestock. Good economic value and application prospects.

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Abstract

公开了一种抗寒及瘦肉型转基因猪及其制备方法，涉及基因工程领域。通过将鼠的解偶联蛋白1基因转到猪的基因组中，获得了一种既能抵抗寒冷又能通过脂肪沉积减少来提高瘦肉率的转基因猪。通过定点单基因的操纵，同时改善猪两个重要生产性状，不仅为大动物基因编辑的应用及其基础研究打下基础，也为育种工作者对家畜进行性状改良带来新的思路。

Description

一种抗寒及瘦肉型转基因猪及其制备方法 技术领域
本发明涉及基因工程领域，具体地说，涉及一种抗寒及瘦肉型转基因猪及其制备方法。
背景技术
在猪的现代育种工作中，减少脂肪过度沉积，增加瘦肉率，提高饲料转化效率是育种工作者一直致力解决的问题。在我国也有较多的脂肪型地方品种，如广西陆川猪，太湖猪等，均需要改良以提高瘦肉率及饲料转化率。
目前猪的重要经济性状的改良主要依赖遗传育种的方法。育种工作已经经历了依赖数量遗传学的常规选育到依赖基因组学和分子生物学的分子标记辅助选择(MAS)和基因型或数量性状位点(QTL)直接选择。虽然常规的选育方法已经在猪生产性状改良上取得了很大的进展，但这一过程需要测定技术和育种方法具有相当的科学性和准确性及可行性，并需要大量人力物力的投入，而且育种周期长。分子育种也面临很大的难题，尤其是面对那些低遗传力的数量性状，鉴定性状相关基因往往要很长时间，花费较高成本，而且许多研究最终都只停留在目标基因的定位上，未进一步走向育种应用。
我国多数瘦肉型商品猪生产的主要途径，大多采用引入外来优良猪种与本地猪种进行杂交获得二元杂种的方法，如直接从国外原产地引进的丹麦长白、英国大约克、美国杜洛克和汉普夏等世界著名瘦肉型猪种，虽然培育出了一批瘦肉型猪新品种(系)，然而，世界一流的品种和育种技术是买不到的，“拿来主义”只能导致我们陷入“引种→退化→再引种→再退化”的被动局面，更不可能使我国畜牧业生产水平与世界发达国家展开真正的竞争。
不仅如此，新出生仔猪的颤抖性产热能力仍不健全，会因较差的产热能力而降低存活率，现代养殖场中，都采用保温灯对仔猪进行保暖以提高 存活率。然而，保温灯的使用无疑增加了生产成本，而对一些寒冷地区却没有配备保温灯的养殖户来说，仔猪存活率势必要受到天气寒冷的影响。
因此，亟需提供一种方法能够既提高猪的瘦肉率及饲料转化率，又能提高猪的抗寒能力。
有研究发现，解偶联蛋白1(UCP1)是一种在棕色脂肪里特异表达，存在于线粒体内膜上的一种蛋白，它能消除线粒体内膜两侧因呼吸链的电子传递而建立的质子浓度差，使这种电化学浓度势能以热量的形式散发，这种产热也被称为非颤抖性产热。然而，猪并不含有棕色脂肪(Jastroch,M.,&Andersson,L.When pigs fly,UCP1makes heat.Molecular Metabolism,4(5),359-362.doi:10.1016/j.molmet.2015.02.00
5)。因此，尚未见到将该基因应用在猪的改良方面的任何报道。
发明内容
为了解决现有技术中存在的问题，本发明的目的是提供一种抗寒及瘦肉型转基因猪及其制备方法。
为了实现本发明目的，本发明的技术方案如下：
第一方面，本发明提供了一种抗寒及瘦肉型转基因猪，其为在脂肪组织中过表达UCP1基因的转基因猪。
所述UCP1基因可选自任意物种有功能的UCP1基因，如人、牛、羊、小鼠等动物。
进一步地，所述UCP1基因为来自鼠的解偶联蛋白1基因，由脂联素启动子特异性启动过表达。
作为优选，所述脂联素启动子为鼠源的脂联素启动子。
本发明供体质粒中的UCP1基因和启动子，由包含鼠脂联素启动子和UCP1编码序列的Pcdna3.1载体酶切得到，其核苷酸序列分别如SEQ ID NO.2和SEQ ID NO.3所示。
第二方面，本发明还提供了前述转基因猪的制备方法，包括如下步骤：
S1、构建含有靶标序列、脂联素启动子和UCP1基因的供体质粒；
S2、构建针对靶标序列的Cas9/gRNA载体；
S3、将构建好的供体质粒和Cas9/gRNA载体对猪胎儿成纤维细胞进行共转染，通过有限稀释法获得单克隆细胞，进一步通过PCR鉴定获得打靶成功的阳性细胞；
S4、将筛选得到的阳性克隆细胞作为核移植供体细胞，离体的卵母细胞为核移植受体细胞，通过核移植技术进行体细胞克隆，获得转基因猪。
所述供体质粒的出发载体可为本领域常规使用的载体，在本发明的一个具体实施方式中，选择pLB载体作为出发载体。
所述供体质粒中的靶标序列位于启动子序列前，UCP1基因位于启动子序列后。所述靶标序列采用麻省理工学院的Zhang Feng教授开发的在线工具CRISPR Design Tool(http://crispr.mit.edu/)来设计并交由Thermo Fisher公司合成，其核苷酸序列如SEQ ID NO.1所示，在猪基因组中位于猪解偶联蛋白1假基因的2号外显子。
所述Cas9/gRNA载体按照本领域常规技术手段制备。包含Cas9的PX330质粒购买自Addgene。首先用BbsⅠ内切酶切割PX330质粒，纯化回收线性化的质粒。接着将gRNA识别序列(即前述的靶标序列)进行变性复性处理，使其由单链核苷酸变成双链寡核苷酸，再用T4DNA连接酶将双链DNA与线性化的PX330质粒进行连接。转化，涂板，摇菌，进一步测序鉴定阳性菌液，大提质粒备用。
第三方面，本发明提供了前述方法在提高转基因猪抗寒能力、和/或提高转基因猪瘦肉率，和/或提高转基因猪饲料转化率方面的应用。
本发明涉及到的原料或试剂均为普通市售产品，涉及到的操作如无特殊说明均为本领域常规操作。
在符合本领域常识的基础上，上述各优选条件，可以相互组合，得到具体实施方式。
本发明的有益效果在于：
本发明通过将鼠的解偶联蛋白1基因转到猪的基因组中获得抵抗寒 冷刺激和脂肪沉积减少瘦肉率增加的转基因猪。
本发明提供了一种既能抵抗寒冷又能通过脂肪沉积减少来提高瘦肉率的转基因猪。本发明通过定点单基因的操纵，同时改善猪两个重要生产性状，不仅为大动物基因编辑的应用及其基础研究打下基础，也为育种工作者对家畜进行性状改良带来新的思路。
附图说明
图1为本发明打靶载体构建及基因敲入鉴定策略。
图2为本发明阳性打靶细胞鉴定。
图3为本发明转基因猪基因型鉴定。
图4为本发明冷冻条件下猪只肛温变化。
图5为本发明冷冻条件下猪只红外照片及体表温度显示。
图6为本发明野生型和转基因猪的屠宰指标检测。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
鼠脂联素启动子启动的UCP1表达载体Pcdna3.1是将鼠脂联素启动子和UCP1编码序列克隆到Pcdna3.1质粒骨架上得到的。序列测定和引物合成由Thermo Fisher公司完成。Taq酶、T4DNA连接酶、内切酶均购自北京NEB公司，体细胞核移植所用试剂均购自Sigma公司。酶切、连接、回收、转化、PCR扩增等常规实验操作步骤参照《分子克隆(第三版)》。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1 转基因猪的制备
按照图1所述的策略进行打靶载体的构建及外源基因的敲入。
1、Cas9/gRNA打靶载体的构建
gRNA的识别序列采用麻省理工学院的Zhang Feng教授开发的在线工具CRISPR Design Tool(http://crispr.mit.edu/)来设计并交由Thermo Fisher公司合成，该序列位于猪解偶联蛋白1假基因的2号外显子。包含Cas9的PX330质粒购买自Addgene。首先用BbsⅠ内切酶切割PX330质粒，纯化回收线性化的质粒。接着将gRNA识别序列进行变性复性处理，使其由单链核苷酸变成双链寡核苷酸，再用T4DNA连接酶将双链DNA与线性化的PX330质粒进行连接。转化，涂板，摇菌，进一步测序鉴定阳性菌液，大提质粒备用。
2、解偶联蛋白1供体质粒的构建
首先，用KpnⅠ和XhoⅠ内切酶对Pcdna3.1质粒进行双酶切，跑胶回收脂联素启动子-UCP1元件。将脂联素启动子-UCP1片段利用pLB零背景快速克隆试剂盒(天根生化科技有限公司)连接到pLB载体上，鉴定阳性质粒，大提备用。最后，用BspEⅠ酶对得到的新质粒进行单酶切，纯化回收，再将带有BspEⅠ酶切位点的gRNA识别序列即靶标序列(Bait sequence)用T4DNA连接酶连到线性化的质粒上，得到供体质粒。
3、UCP1转基因成纤维细胞的获得
3.1猪胎儿成纤维细胞的建立
1)取妊娠35天的巴马母猪，处死后取子宫，1h内运回实验室，从子宫内取出胎儿，用含抗生素的DPBS清洗，转移到超净台内，用眼科剪去掉头、四肢和内脏。将剩余部分用DPBS反复冲洗，直至干净。在100mm培养皿中用眼科剪将剩余部分尽量剪碎。
2)将剪碎的组织转移到新的100mm培养皿中，加10mL消化液在37℃，5％CO 2培养箱中消化4-6h。消化液的成分：DMEM高糖培养基(Gibco)添加15％胎牛血清(Hyclone)，0.032％胶原酶Ⅳ(Sigma)，25Kunitz units/mL DNase Ⅰ(Sigma)和40μg/mL庆大霉素(Sigma)。消 化完毕后，收集消化产物，置于离心管中3000rpm离心10min，弃上清，用培养基重悬后再离心。最后将重悬后的组织铺在25cm 2的培养皿中。培养基成分：DMEM高糖培养基(Gibco)添加15％胎牛血清(Hyclone)和40μg/mL庆大霉素(Sigma)。
3)待细胞生长至80％汇合度时，进行传代培养或冷冻保存。
3.2打靶载体转染胎儿成纤维细胞
转染前两天复苏胎儿成纤维细胞至25cm2培养皿中，待细胞长至70％左右时即可进行转染，转染前4h更换培养基。用0.25％的胰蛋白酶(Invitrogen)消化细胞，用电转液重悬，向重悬细胞中加入Cas9/gRNA质粒和供体质粒。用核电转仪(Nucleofector 2b Device，Lonza)进行转染，采用U-023程序。转染后的细胞悬液转入60mm培养皿中，培养48h。
3.3细胞筛选及鉴定
用0.25％的胰蛋白酶(Invitrogen)消化细胞，终止消化反应后用手持式自动细胞计数仪(Millpore)进行细胞密度检测，根据细胞密度使用有限稀释法，将细胞接种到96孔板中，每个孔添加100ul培养基，并使每100ul培养基中仅含一个细胞。3-4天后换液，待细胞在96孔板中长满时传至24孔板中，24孔板中长满的细胞一部分传至6孔板，另一小部分在24孔板中继续培养，用于基因型鉴定。最后共获得26个细胞克隆，进行PCR鉴定，发现同时在5’连接端和3’连接端扩出条带的为转基因阳性克隆，其中8、14、17号为阳性克隆(图2)，用于后续实验。
4、核移植胚胎及克隆猪的制备
4.1猪卵母细胞的成熟培养：
从屠宰场收集猪的卵巢放于37℃含有青霉素和链霉素的生理盐水的保温瓶中，1小时内运送回实验室。用生理盐水洗卵巢3-5遍后，抽取卵巢表面直径为3～8mm的卵泡，将含有卵母细胞的卵泡液收集在50mL离心管中，用TL-HEPES洗卵液洗涤3遍，在体视显微镜下挑取含有三层以上卵丘细胞、致密且胞质均匀的卵丘-卵母细胞复合物(COCs)，移入 体外成熟培养液中，在39.0℃，5％CO 2条件下培养42～44h。
4.2重构胚胎的获得：
将体外成熟培养42～44h的卵母细胞用0.1％透明质酸酶(购自Sigma公司)消化，去除卵母细胞周围的卵丘细胞，在体视显微镜下挑取胞质均匀，卵周隙明显并已排出第一极体的卵母细胞，即MII期卵母细胞，置于显微操作滴中，用固定管固定成熟卵母细胞，使极体位于3点钟方向，用外径20μm的去核针将第一极体和周围约1/8的细胞质(包含细胞核)吸除出去；将UCP1转基因的成纤维细胞注入去核的卵母细胞卵周隙中，使供体细胞与卵母细胞膜接触；然后利用电激活方法(BTX Electro-cell Manipulator 200)使卵母细胞与供体细胞融合，获得重构胚胎。将重构胚胎置于PZM3液中，在39.0℃，5％CO 2条件下培养14～16小时，然后进行胚胎移植。
4.3胚胎移植：
将体外培养14-16小时的重构胚胎移入同期发情的假孕母猪输卵管中，平均每头母猪移植约180枚胚胎。28天后通过超声波检测母猪是否怀孕。如果怀孕，则每两周超声波监测一次怀孕情况。本发明得到3头受孕母猪，顺利产下12头小猪。
5、UCP1基因敲入猪的鉴定
猪出生后，取耳朵组织，用基因组DNA提取试剂盒(天根生化科技有限公司)提取DNA，进行PCR鉴定(图3)。
实施例2 仔猪抗寒能力的检测
将1月龄转基因猪和野生型猪放在4°冰箱中，冷冻4h。从0h开始，每隔1h用电子温度计(天津今明仪器公司)测量猪肛温(图4)，由图4可知，冷刺激1h-4h之间，转基因猪的肛温显著高于野生型猪。同时用红外相机(FLIR)对猪照相，记录猪体表温度变化(图5)。由图5可知，冷刺激后，转基因猪体表红外显示颜色要明显强于野生型，即白色线标记区域内的温度转基因猪高于野生型的。
实施例3 屠宰指标检测
待猪到6月龄时进行屠宰实验。屠宰前24h禁食。宰前称重，麻醉放血处死。去头，蹄，尾和内脏(保留肾脏和板油)，称重，记为胴体重。胴体重占宰前重的比例即为屠宰率。将猪胴体从中间分开，针对左侧胴体，分离瘦肉，脂肪，皮和骨，分别称重，计算各个部分占总和的比例(见图6)。由图6可知，野生型和转基因猪的屠宰率无差异，转基因猪的瘦肉率显著高于，脂肪率显著低于野生型。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种抗寒及瘦肉型转基因猪及其制备方法。本发明通过将鼠的解偶联蛋白1基因转到猪的基因组中，获得了一种既能抵抗寒冷又能通过脂肪沉积减少来提高瘦肉率的转基因猪。本发明提供的转基因猪既能抵抗寒冷又能通过脂肪沉积减少来提高瘦肉率。本发明通过定点单基因的操纵，同时改善猪两个重要生产性状，不仅为大动物基因编辑的应用及其基础研究打下基础，也为育种工作者对家畜进行性状改良带来新的思路，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种抗寒及瘦肉型转基因猪，其特征在于，其为在脂肪组织中过表达UCP1基因的转基因猪。
  2. 根据权利要求1所述的转基因猪，其特征在于，所述UCP1基因为来自鼠的解偶联蛋白1基因。
  3. 根据权利要求1或2所述的转基因猪，其特征在于，所述UCP1基因由脂联素启动子特异性启动过表达。
  4. 根据权利要求3所述的转基因猪，其特征在于，所述脂联素启动子为鼠源的脂联素启动子。
  5. 权利要求1～4任一项所述的转基因猪的制备方法，其特征在于，包括如下步骤：
    S1、构建含有靶标序列、脂联素启动子和UCP1基因的供体质粒；
    S2、构建针对靶标序列的Cas9/gRNA载体；
    S3、将构建好的供体质粒和Cas9/gRNA载体对猪胎儿成纤维细胞进行共转染，通过有限稀释法获得单克隆细胞，进一步通过PCR鉴定获得打靶成功的阳性细胞；
    S4、将筛选得到的阳性克隆细胞作为核移植供体细胞，离体的卵母细胞为核移植受体细胞，通过核移植技术进行体细胞克隆，获得转基因猪。
  6. 根据权利要求5所述的方法，其特征在于，所述供体质粒的出发载体为pLB载体。
  7. 根据权利要求6所述的方法，其特征在于，所述靶标序列的核苷酸序列如SEQ ID NO.1所示。
  8. 权利要求5～7任一项所述的方法在提高转基因猪抗寒能力方面的应用。
  9. 权利要求5～7任一项所述的方法在提高转基因猪瘦肉率方面的应用。
  10. 权利要求5～7任一项所述的方法在提高转基因猪饲料转化率方 面的应用。
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