Advances on transgenic and gene-edited pigs
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摘要:
猪是重要的农业经济动物，其在遗传背景、解剖学、生理病理学、营养代谢和疾病特征等方面与人类高度相似，是农业生产性状改良、人类疾病动物模型和异种器官移植研究的重要对象。随着ZFN、TALEN和CRISPR等基因编辑技术的出现，转基因或基因编辑猪的研究取得快速发展。本文将结合当前我国转基因及基因编辑猪的研究现状，对基因编辑技术在农业生产性状改良如提高产肉量、改善肉质、提高抗病能力和建立人类疾病模型、动物生物反应器、异种器官移植等方面进行综述，以期为猪遗传改良和医学研究提供参考。
Abstract:Pigs are important agricultural animals and highly similar to humans in terms of genetic background, anatomy, physiological pathology, nutrient metabolism and disease characteristics. Pigs are intensively studied for the improvement of production traits, establishment of animal models for human diseases and xenotransplantation. With the emergence of gene editing technologies, such as ZFN, TALEN and CRISPR, the research on transgenic or gene-edited pigs has achieved a rapid development. This review summarizes the current research advance of transgenic and gene-edited pigs in China. We also review the applications of gene editing technology in agricultural area, such as increasing meat production, improving meat quality, and resisting disease, as well as in biomedical study such as establishing human disease models, animal bioreactors and xenotransplantation for references of pig genetic improvement and medical research.
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Keywords:
- transgenic pig /
- gene-edited pig /
- genetic improvement /
- disease model /
- bioreactor /
- xenotransplantation
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自动驾驶技术研发中，行驶车辆的水平位置(经度、纬度)和航向角度是两大关键信息，主要为车辆控制系统的横向控制(转向盘控制)和纵向控制(制动、加速控制)提供参考数据，满足自动驾驶车辆定位导航的综合需求[1]。自动驾驶技术对车辆航向角测量精度要求非常高。对高速行驶的汽车而言，航向角轻微偏差都会导致汽车偏离原来的行驶路线。对农业机械而言，虽然对农机作业速度要求不高，但对导航作业精度要求很高(特别是播种时需要达到cm级)，航向角的轻微偏差都会对导航作业精度产生很大影响。因此，提高车辆航向角的测量精度是非常必要的。车辆航向角测量方法主要有磁阻传感器法、双天线全球导航卫星系统(Global navigation satellite system，GNSS)定位定向法、单天线GNSS定位定向法和陀螺仪测航向法等。磁阻传感器受周围磁场环境影响大，精度和可靠性不高，在高压线等有磁场干扰的作业环境下容易受到影响[2-5]；双天线GNSS定位系统测航向，虽然测量精度高，但动态响应特性差，成本高[6-7]；单天线GNSS定位系统可输出航向角度信息，但是随机噪声大，且速度越低，噪声越大[8-11]；陀螺仪测航向一方面需要航向角度初始化，另一方面随机漂移误差会出现累积发散现象[ 12-15]。上述几种测量方法测得的航向角，均不能很好地满足车辆自动驾驶的精度要求。
本文采用单天线GNSS定位和微电子机械系统(Micro electro mechanical system, MEMS)陀螺仪相结合的方式，通过融合算法实现车辆航向角的测量。提出基于卡尔曼滤波器的车辆航向角估计模型，把实时动态–全球导航卫星系统(Real time kinematic-GNSS，RTK-GNSS)测量出来的经纬度和高程经过高斯投影变换为导航平面坐标，与陀螺仪测量的车辆角速度经过积分得出的航向角做融合处理，得到更为精准的航向角。该方法克服了GNSS因更新频率低出现的数据延迟和MEMS陀螺仪因随机漂移引起的累积误差等问题，不仅能够得到更为精准的航向角数据，还能保证良好的实时性。
1. 传感器组合
本文测量车辆航向角采用的导航传感器主要有Trimble®BD970 GNSS嵌入式板卡和内置于Xsens MTi-300微型姿态参考系统的MEMS陀螺仪。
1.1 Trimble®BD970 GNSS嵌入式板卡
Trimble®BD970 GNSS嵌入式板卡是一款紧凑型的多星接收机板卡，专为满足各种精确到cm级的定位精度应用需求而设计。系统模块不仅支持GPS L1/L2、L2C、L5，而且支持GLONASS L1/L2 信号在内的各种卫星信号。该板卡易于集成且坚固可靠，支持因特网、USB、RS232 和CAN 等多种接口，串口输出波特率最高达115 200 bps，可实现高达50 Hz的原始测量与定位输出。基准站输出支持CMR、CMR+、RTCM 2.1、2.2、2.3、3.0、3.1等协议格式；定位数据输出支持ASCII：NMEA-0183 GSV、AVR、RMC、HDT、VGK、VHD、ROT、GGK、GGA、GSA、ZDA、VTG、GST、PJT、PJK、BPQ、GLL、GRS、GBS以及二进制：TrimbleGSOF。低延时RTK定位模式的水平定位精度可达±(8 mm+1 ppm)RMS，垂直定位精度可达±(15 mm+1 ppm)RMS，延迟时间小于20 ms，最大输出频率50 Hz。GNSS板卡物理特性如下，尺寸：100 mm×60 mm×11.6 mm；电源：3.3 V DC(−3%~5%)；典型功耗：1.4 W (L1/L2 GPS)或1.5 W (L1/L2 GPS和G1/G2 GLONASS)；质量：62 g；连接器I/O：24排针转接口和6排针转接口；天线：MMCX插座；工作温度：−40~75 ℃；储存温度：−55~85 ℃；振动限值：随机8 g RMS。
1.2 Xsens MTi-300微型姿态航向参考系统
Xsens Technologies B.V.公司研发的MTi-300微型姿态航向参考系统内部包括：3D速率陀螺、3D加速度计和3D磁场感应计。运行于DSP上的卡尔曼滤波算法融合上述传感器信息，给出运动载体的精确3D姿态角度[8]。系统通过RS232接口按设定格式输出3D姿态角度。MTi内置的3D速率陀螺的测量范围可达±300°/s，零偏稳定性为1°/s，随机游走系数0.05°/(s·Hz)，校准误差0.1°，带宽40 Hz，A/D分辨率16位，更新速率最大为120 Hz。本文利用3D速率陀螺中的Z轴陀螺实现车辆航向角的角速率累积测量。
2. 航向角估计算法设计
2.1 GNSS定位数据预处理
GNSS定位数据预处理主要将Trimble®BD970 GNSS板卡测量得到的WGS-84大地坐标系的经度、纬度和高程向大地导航坐标系转换，通过高斯投影将WGS-84大地坐标转换为与WGS-84椭球对应的高斯平面坐标，这种转换是为了使GNSS板卡输出的WGS-84大地坐标定位数据能够用于车辆的导航控制系统。
本文使用的Gauss-Kruger投影坐标系的主要参数[12]包括：中央经线为114.000 000 (3度带)；水平偏移量为500 km；地理坐标系为GCS_WGS_1984；大地参照系为D_WGS_1984；参考椭球体为WGS_1984；椭球长轴为6 378 137.000 000；椭球扁率为0.003 352 810 7。
2.2 卡尔曼滤波器设计
设定k时刻车辆本体的真实航向角度为ψk'，车辆本体的真实前进速度是vk'，则
$$ \psi _{{k}}' = {\psi _{{k}}} + {\varepsilon _{{{\psi k}}}} + {\xi _{{{\psi k}}}}\text{，} $$ (1) $$ v_{{k}}' = {v_{{k}}} + {\xi _{{{vk}}}}\text{，} $$ (2) 式中，ψk、vk分别指航向角度、前进速度的测量值，εψk指航向角度的测量偏差值，ξψk、ξvk分别指航向角度和前进速度的随机测量误差。
基于航位推算原理，建立车辆导航控制点在2D平面坐标系下的运动方程：
$$ {x_{{{ck}}}} = {x_{{{ck}} - 1}} + v_{{k}}' \cos \theta _{{k}}' {\rm{d}}t\text{，} $$ (3) $$ {y_{{{ck}}}} = {y_{{{ck}} - 1}} + v_{{k}}' \sin \theta _{{k}}' {\rm{d}}t\text{，} $$ (4) 式中，xck、yck为k时刻车辆本体的高斯投影平面坐标，xck−1、yck−1为k−1时刻车辆本体的高斯投影平面坐标，dt为航位推算的时间间隔。
将(1)和(2)式代入上述表达式，得到：
$$ \begin{split} {x_{{ck}}} = & {x_{{{ck}} - 1}} + {v_{{k}}}\cos {\psi _{{k}}}{\rm{d}}t + {\varepsilon _{{{\psi k}}}}\cos {\psi _{{k}}}{\rm{d}}t - \\ &{v_{{k}}}{\varepsilon _{{{\psi k}}}}\sin {\psi _{{k}}}{\rm{d}}t\text{，} \end{split} $$ (5) $$\begin{split} {y_{ck}} =& {y_{{{ck}} - 1}} + {v_{{k}}}{\rm{sin}}{\psi _k}{\rm{d}}t + {\varepsilon _{\psi {{k}}}}\sin {\psi _{{k}}}{\rm{d}}t + \\ &{v_k}{\varepsilon _{\psi k}}\cos {\psi _k}{\rm{d}}t\text{。} \end{split} $$ (6) 将上述等式以卡尔曼滤波器状态转移方程的形式表示为：
$$ {{\boldsymbol{X}}_{{k}}} = {{\boldsymbol{A}}_{{k}}}{{\boldsymbol{X}}_{{{k}} - 1}} + {{\boldsymbol{b}}_{{k}}} + {{\boldsymbol{u}}_{{k}}}\text{，} $$ (7) 式中，Xk=[xck，yck，εψk]，表示k时刻的状态空间向量；Xk−1=[xck−1，yck−1，εψk−1]，表示k−1时刻的状态空间向量；
$$ {{\boldsymbol{A}}_{{k}}} = \left[ {\begin{array}{*{20}{c}} 1&0&{\left( {{\rm{cos}}{\psi _{{k}}} - {v_{{k}}}\sin {\psi _{{k}}}} \right){\rm{d}}t}\\ 0&1&{\left( {{\rm{sin}}{\psi _{{k}}} + {v_{{k}}}\cos {\psi _{{k}}}} \right){\rm{d}}t}\\ 0&0&1 \end{array}} \right]\text{，} $$ 是k时刻状态转移矩阵，由陀螺仪累积航向角度和前进速度的测量值实时更新；
$$ {{\boldsymbol{b}}_{{k}}} = \left[ {\begin{array}{*{20}{c}} {{v_{{k}}}\cos {\psi _{{k}}}{\rm{d}}t}\\ {{v_{{k}}}\sin {\psi _{{k}}}{\rm{d}}t}\\ 0 \end{array}} \right]\text{，} $$ $$ {{\boldsymbol{u}}_{{k}}} = \left[ {0,\;\;0,\;\;{\xi _{\psi {{k}}}}} \right]\text{，} $$ 是状态转移方程的白噪声序列；系统过程噪声协方差矩阵为Qk，表示状态转移方程的误差大小，本文中Qk设定为常数矩阵，在仿真和试验过程中整定矩阵参数。
以GNSS天线在大地导航坐标系下的定位坐标作为观测向量，得到卡尔曼滤波器的测量方程如下：
$$ {{\boldsymbol{Z}}_{{{gk}}}} = {H_k}{X_k} + {{\boldsymbol{\nu}} _k} $$ (8) 式中，
$$ {{\boldsymbol{Z}}_{{{gk}}}} = \left[ {\begin{array}{*{20}{c}} {{x_{{{gk}}}}}\\ {{y_{{{gk}}}}} \end{array}} \right]\text{，} $$ $$ {{\boldsymbol{H}}_{{k}}} = \left[ {\begin{array}{*{20}{c}} 1&0&0\\ 0&1&0 \end{array}} \right]\text{，} $$ $$ {{\boldsymbol{\nu}} _{{k}}} = \left[ {\begin{array}{*{20}{c}} {{\xi _{{{gxk}}}}}\\ {{\xi _{{{gyk}}}}} \end{array}} \right]\text{，} $$ 式中，xgk、ygk为GNSS天线处的定位坐标，Hk为卡尔曼滤波器k时刻的测量矩阵，ξgxk、ξgyk为OEM GNSS板卡定位在水平面坐标系下的随机定位误差。
测量向量的噪声方差矩阵为：
$$ {{\boldsymbol{R}}_k} = \left[ {\begin{array}{*{20}{c}} {{r_{xk}}^2}&0\\ 0&{{r_{yk}}^2} \end{array}} \right]\text{，} $$ (9) 式中，rxk2、ryk2分别为ξgxk、ξgyk的方差统计值。
综合上述推导，采用线性离散卡尔曼滤波器的递归差分方程进行状态向量预测和测量向量校正：
预测方程组为：
$$ {\hat x_k} = {{\boldsymbol{A}}_k}{\hat x_{k-1}} + {{\boldsymbol{b}}_k}\text{，} $$ (10) $$ {{\boldsymbol{P}}_k} = {{\boldsymbol{A}}_k}{{\boldsymbol{P}}_{k - 1}}{{\boldsymbol{A}}_k}^T + {{\boldsymbol{Q}}_{k - 1}}\text{，} $$ (11) 式中，
${\hat x_k} $ 表示k时刻的预测结果，${\hat x_{k-1}}$ 表示k−1时刻的预测结果，Ak表示状态转移矩阵，${\boldsymbol{A}}_k^T$ 代表Ak的转置，Pk对应${\hat x_k} $ 在k时刻的系统过程噪声方差预测值，Pk−1对应${\hat x_{k-1}}$ 在k−1时刻的系统过程噪声方差预测值，Qk−1为k−1时刻的系统过程协方差。校正方程组为：
$$ {{\boldsymbol{K}}_k} = {{\boldsymbol{P}}_k} {{\boldsymbol{H}}_k}^T{({{\boldsymbol{H}}_k}{{\boldsymbol{P}}_k} {{\boldsymbol{H}}_k}^T + {{\boldsymbol{R}}_k})^{ - 1}}\text{，} $$ (12) $$ {\hat x'_k} = {\hat x_k} + {{\boldsymbol{K}}_k}\left( {{{\boldsymbol{Z}}_{gk}} - {{\boldsymbol{K}}_k}{{\hat x}_k}} \right)\text{，} $$ (13) $$ {{\boldsymbol{P}}_k} = \left( {I - {{\boldsymbol{K}}_k}{{\boldsymbol{H}}_k}} \right){{\boldsymbol{P}}_k} \text{。} $$ (14) 式中，Kk为k时刻的卡尔曼滤波增益，
${\boldsymbol{H}}_k^T$ 为Hk的转置，${\hat x'_k}$ 是k时刻最优化估计值，Zgk是k时刻的测量更新值，I为单位矩阵。3. 结果与分析
采用GNSS板卡和MEMS陀螺仪在轮式拖拉机平台上进行原始数据采集。GNSS天线安装于车辆后轮轴中心点的正上方。内置MEMS陀螺仪的MTi尽可能安装于车辆质心位置处，以减少车辆颠簸晃动对MTi的干扰。
轮式拖拉机的行驶路线分直线型和S型2种情况。用C++编程语言开发卡尔曼滤波器和原始数据仿真测试的程序。最后将测得的数据以文本文件的方式导入Matlab程序中，测得的曲线图如图1、2所示。
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图 1 直线行驶时拖拉机航向角的估计结果Figure 1. The estimation result of heading angle of the tractor running in straight trajectory![]()
图 2 S型路线行驶时拖拉机航向角的估计结果Figure 2. The estimation result of heading angle of the tractor running in S type trajectory图1为拖拉机直线行驶时，GNSS、陀螺仪和卡尔曼滤波融合后得到的3条航向角度对比曲线。GNSS航向误差幅度超过5°，陀螺仪累积航向的偏移在300 s左右超过2°，融合后的航向角度都在38°左右，偏移不超过1°，较原始GNSS航向角度的精度提高80%以上。
图2为拖拉机以S型轨迹行驶时，GNSS、陀螺仪和卡尔曼滤波融合后得到的3条航向角度对比曲线。融合后的航向角度可以跟踪拖拉机180°换向的转弯动作，曲线既保持了GNSS航向的整体变化趋势，也较GNSS和陀螺仪所得结果更为平滑，符合拖拉机实际运动状态。
从图1、2中可看出，未经处理的陀螺仪累积航向角度和GNSS定位测量的航向角度有较大波动，经卡尔曼滤波融合后，有效抑制了陀螺仪累积航向的发散，减少了零偏和随机漂移带来的误差。融合后的航向角度曲线既保持了GNSS航向的整体变化趋势，也保持了陀螺仪航向的细部变化趋势，且较GNSS和陀螺仪所得曲线更为平滑。
4. 结论
本文采用卡尔曼滤波器对RTK-GNSS、MEMS陀螺仪所得的拖拉机航向角进行融合处理，得出了更为精确的航向角融合估计结果，仿真测试结果表明本文所用方法可用于在线测量拖拉机航向角。
RTK-GNSS航向角和MEMS陀螺仪累积航向角在采样频率方面，分别属于低频型和高频型；在误差特性方面，分别为零均值随机误差和偏移型缓变误差。2种传感器互补性强，研究结果表明多传感器融合的方法能够很好地弥补这2种传感器单独测量数据时存在的噪声误差。
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[1] WANG J, LIU M, ZHAO L, et al. Disabling of nephrogenesis in porcine embryos via CRISPR/Cas9-mediated SIX1 and SIX4 gene targeting[J]. Xenotransplantation, 2019, 26(3): e12484. doi: 10.1111/xen.2019.26.issue-3
[2] YAN S, TU Z, LIU Z, et al. A Huntingtin knockin pig model recapitulates features of selective neurodegeneration in Huntington's disease[J]. Cell, 2018, 173(4): 989-1002. doi: 10.1016/j.cell.2018.03.005
[3] HAI T, GUO W, YAO J, et al. Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis[J]. Hum Genet, 2017, 136(11/12): 1463-1475. doi: 10.1007/s00439-017-1851-2
[4] HSU P D, LANDER E S, ZHANG F. Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157(6): 1262-1278. doi: 10.1016/j.cell.2014.05.010
[5] NISHIMASU H, RAN F A, HSU P D, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA[J]. Cell, 2014, 156(5): 935-949. doi: 10.1016/j.cell.2014.02.001
[6] DOUDNA J A, CHARPENTIER E. The new frontier of genome engineering with CRISPR-Cas9[J]. Science, 2014, 346(6213): 1258096. doi: 10.1126/science.1258096
[7] RAN F A, HSU P D, LIN C Y, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity[J]. Cell, 2013, 154(6): 1380-1389. doi: 10.1016/j.cell.2013.08.021
[8] QI L S, LARSON M H, GILBERT L A, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression[J]. Cell, 2013, 152(5): 1173-1183. doi: 10.1016/j.cell.2013.02.022
[9] 吴添文, 齐传翔, 李训碧, 等. 基因组编辑猪的研究现状及展望[J]. 农业生物技术学报, 2017, 25(5): 781-787. [10] ZHANG H X, ZHANG Y, YIN H. Genome editing with mRNA encoding ZFN, TALEN, and Cas9[J]. Mol Ther, 2019, 27(4): 735-746. doi: 10.1016/j.ymthe.2019.01.014
[11] TOWNSEND J A, WRIGHT D A, WINFREY R J, et al. High-frequency modification of plant genes using engineered zinc-finger nucleases[J]. Nature, 2009, 459(7245): 442-445. doi: 10.1038/nature07845
[12] LOMBARDO A, GENOVESE P, BEAUSEJOUR C M, et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery[J]. Nat Biotechnol, 2007, 25(11): 1298-1306. doi: 10.1038/nbt1353
[13] DURAI S, MANI M, KANDAVELOU K, et al. Zinc finger nucleases: Custom-designed molecular scissors for genome engineering of plant and mammalian cells[J]. Nucleic Acids Res, 2005, 33(18): 5978-5990. doi: 10.1093/nar/gki912
[14] CHU C, YANG Z, YANG J, et al. Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases[J]. BMC Biotechnol, 2019, 19: 7. doi: 10.1186/s12896-018-0494-2
[15] GUILINGER J P, PATTANAYAK V, REYON D, et al. Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity[J]. Nat Methods, 2014, 11(4): 429-435. doi: 10.1038/nmeth.2845
[16] CERMAK T, DOYLE E L, CHRISTIAN M, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting[J]. Nucleic Acids Res, 2011, 39(12): e82. doi: 10.1093/nar/gkr218
[17] LI T, HUANG S, ZHAO X, et al. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes[J]. Nucleic Acids Res, 2011, 39(14): 6315-6325. doi: 10.1093/nar/gkr188
[18] HIRANO S, NISHIMASU H, ISHITANI R, et al. Structural basis for the altered PAM specificities of engineered CRISPR-Cas9[J]. Mol Cell, 2016, 61(6): 886-894. doi: 10.1016/j.molcel.2016.02.018
[19] HU J H, MILLER S M, GEURTS M H, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity[J]. Nature, 2018, 556(7699): 57-63. doi: 10.1038/nature26155
[20] XU S, CAO S, ZOU B, et al. An alternative novel tool for DNA editing without target sequence limitation: The structure-guided nuclease[J]. Genome Biol, 2016, 17(1): 186. doi: 10.1186/s13059-016-1038-5
[21] SWARTS D C, VAN DER OOST J, JINEK M. Structural basis for guide RNA processing and seed-dependent DNA targeting by CRISPR-Cas12a[J]. Mol Cell, 2017, 66(2): 221-233. doi: 10.1016/j.molcel.2017.03.016
[22] KIM E, KOO T, PARK S W, et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni[J]. Nat Commun, 2017, 8: 14500. doi: 10.1038/ncomms14500
[23] RAN F A, CONG L, YAN W X, et al. In vivo genome editing using Staphylococcus aureus Cas9[J]. Nature, 2015, 520(7546): 186-191. doi: 10.1038/nature14299
[24] HARRINGTON L B, BURSTEIN D, CHEN J S, et al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes[J]. Science, 2018, 362(6416): 839-842. doi: 10.1126/science.aav4294
[25] CRUZ-GARCIA A, LOPEZ-SAAVEDRA A, HUERTAS P. BRCA1 accelerates CtIP-mediated DNA-end resection[J]. Cell Rep, 2014, 9(2): 451-459. doi: 10.1016/j.celrep.2014.08.076
[26] HEYER W D, EHMSEN K T, LIU J. Regulation of homologous recombination in eukaryotes[J]. Annu Rev Genet, 2010, 44: 113-139. doi: 10.1146/annurev-genet-051710-150955
[27] CHEN Y, ZHENG Y, KANG Y, et al. Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9[J]. Hum Mol Genet, 2015, 24(13): 3764-3774. doi: 10.1093/hmg/ddv120
[28] YANG H, WU Z. Genome editing of pigs for agriculture and biomedicine[J]. Front Genet, 2018, 9: 360. doi: 10.3389/fgene.2018.00360
[29] PIERZCHALA M, PAREEK C S, URBANSKI P, et al. Study of the differential transcription in liver of growth hormone receptor (GHR), insulin-like growth factors (IGF1, IGF2) and insulin-like growth factor receptor (IGF1R) genes at different postnatal developmental ages in pig breeds[J]. Mol Biol Rep, 2012, 39(3): 3055-3066. doi: 10.1007/s11033-011-1068-8
[30] JEON J T, CARLBORG O, TORNSTEN A, et al. A paternally expressed QTL affecting skeletal and cardiac muscle mass in pigs maps to the IGF2 locus[J]. Nat Genet, 1999, 21(2): 157-158. doi: 10.1038/5938
[31] XIANG G, REN J, HAI T, et al. Editing porcine IGF2 regulatory element improved meat production in Chinese Bama pigs[J]. Cell Mol Life Sci, 2018, 75(24): 4619-4628. doi: 10.1007/s00018-018-2917-6
[32] LIU X, LIU H, WANG M, et al. Disruption of the ZBED6 binding site in intron 3 of IGF2 by CRISPR/Cas9 leads to enhanced muscle development in Liang Guang Small Spotted pigs[J]. Transgenic Res, 2019, 28(1): 141-150. doi: 10.1007/s11248-018-0107-9
[33] QIAN L, TANG M, YANG J, et al. Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs[J]. Sci Rep, 2015, 5: 14435. doi: 10.1038/srep14435
[34] WANG K, OUYANG H, XIE Z, et al. Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system[J]. Sci Rep, 2015, 5: 16623. doi: 10.1038/srep16623
[35] ZHENG Q, LIN J, HUANG J, et al. Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity[J]. Proc Natl Acad Sci USA, 2017, 114(45): E9474-E9482. doi: 10.1073/pnas.1707853114
[36] SAEKI K, MATSUMOTO K, KINOSHITA M, et al. Functional expression of a Delta12 fatty acid desaturase gene from spinach in transgenic pigs[J]. Proc Natl Acad Sci USA, 2004, 101(17): 6361-6366. doi: 10.1073/pnas.0308111101
[37] JIANG W, OKEN H, FIUZAT M, et al. Plasma omega-3 polyunsaturated fatty acids and survival in patients with chronic heart failure and major depressive disorder[J]. J Cardiovasc Transl Res, 2012, 5(1): 92-99. doi: 10.1007/s12265-011-9325-8
[38] KANG J X. The omega-6/omega-3 fatty acid ratio in chronic diseases: Animal models and molecular aspects[J]. World Rev Nutr Diet, 2011, 102: 22-29. doi: 10.1159/000327787
[39] LAI L, KANG J X, LI R, et al. Generation of cloned transgenic pigs rich in omega-3 fatty acids[J]. Nat Biotechnol, 2006, 24(4): 435-436. doi: 10.1038/nbt1198
[40] ZHANG P, ZHANG Y, DOU H, et al. Handmade cloned transgenic piglets expressing the nematode fat-1 gene[J]. Cell Reprogram, 2012, 14(3): 258-266. doi: 10.1089/cell.2011.0073
[41] LI M, OUYANG H, YUAN H, et al. Site-specific fat-1 knock-in enables significant decrease of n-6PUFAs/n-3PUFAs ratio in pigs[J]. G3-Genes Genom Genet, 2018, 8(5): 1747-1754.
[42] TANG F, YANG X, LIU D, et al. Co-expression of fat1 and fat2 in transgenic pigs promotes synthesis of polyunsaturated fatty acids[J/OL]. Transgenic Res, 2019. http://doi.org/10.1007/s11248-019-00127-4
[43] ZHANG L, ZHOU Y, WU W, et al. Skeletal muscle-specific overexpression of PGC-1α induces fiber-type conversion through enhanced mitochondrial respiration and fatty acid oxidation in mice and pigs[J]. Int J Biol Sci, 2017, 13(9): 1152-1162. doi: 10.7150/ijbs.20132
[44] YING F, ZHANG L, BU G, et al. Muscle fiber-type conversion in the transgenic pigs with overexpression of PGC1α gene in muscle[J]. Biochem Biophys Res Commun, 2016, 480(4): 669-674. doi: 10.1016/j.bbrc.2016.10.113
[45] ZHANG X, LI Z, YANG H, et al. Novel transgenic pigs with enhanced growth and reduced environmental impact[J]. Elife, 2018, 7: e34286. doi: 10.7554/eLife.34286
[46] GOLOVAN S P, MEIDINGER R G, AJAKAIYE A, et al. Pigs expressing salivary phytase produce low-phosphorus manure[J]. Nat Biotechnol, 2001, 19(8): 741-745. doi: 10.1038/90788
[47] LIN Y S, YANG C C, HSU C C, et al. Establishment of a novel, eco-friendly transgenic pig model using porcine pancreatic amylase promoter-driven fungal cellulase transgenes[J]. Transgenic Res, 2015, 24(1): 61-71. doi: 10.1007/s11248-014-9817-9
[48] HUANG M, LI Z, HUANG X, et al. Co-expression of two fibrolytic enzyme genes in CHO cells and transgenic mice[J]. Transgenic Res, 2013, 22(4): 779-790. doi: 10.1007/s11248-012-9681-4
[49] ZHANG M, CAI G, ZHENG E, et al. Transgenic pigs expressing beta-xylanase in the parotid gland improve nutrient utilization[J]. Transgenic Res, 2019, 28(2): 189-198. doi: 10.1007/s11248-019-00110-z
[50] HU S, QIAO J, FU Q, et al. Transgenic shRNA pigs reduce susceptibility to foot and mouth disease virus infection[J]. Elife, 2015, 4: e6951.
[51] XIE Z, PANG D, YUAN H, et al. Genetically modified pigs are protected from classical swine fever virus[J]. PLoS Pathog, 2018, 14(12): e1007193. doi: 10.1371/journal.ppat.1007193
[52] YAN Q, YANG H, YANG D, et al. Production of transgenic pigs over-expressing the antiviral gene Mx1[J]. Cell Regen (Lond), 2014, 3(1): 11.
[53] 江戈龙, 高文超, 刘德武, 等. 母源抗体对感染PCV2仔猪体内病毒变化和免疫反应的影响[J]. 中国兽医学报, 2017, 37(3): 398-403. [54] NIU P, SHABIR N, KHATUN A, et al. Effect of polymorphisms in the GBP1, Mx1 and CD163 genes on host responses to PRRSV infection in pigs[J]. Vet Microbiol, 2016, 182: 187-195. doi: 10.1016/j.vetmic.2015.11.010
[55] ZHU L, SONG H, ZHANG X, et al. Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus- and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors[J]. Virol J, 2014, 11: 225. doi: 10.1186/s12985-014-0225-9
[56] WHITWORTH K M, ROWLAND R R, EWEN C L, et al. Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus[J]. Nat Biotechnol, 2016, 34(1): 20-22. doi: 10.1038/nbt.3434
[57] PRATHER R S, ROWLAND R R, EWEN C, et al. An intact sialoadhesin (Sn/SIGLEC1/CD169) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus[J]. J Virol, 2013, 87(17): 9538-9546. doi: 10.1128/JVI.00177-13
[58] WHITWORTH K M, LEE K, BENNE J A, et al. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos[J]. Biol Reprod, 2014, 91(3): 78.
[59] BURKARD C, LILLICO S G, REID E, et al. Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function[J]. PLoS Pathog, 2017, 13(2): e1006206. doi: 10.1371/journal.ppat.1006206
[60] BURKARD C, OPRIESSNIG T, MILEHAM A J, et al. Pigs lacking the scavenger receptor cysteine-rich domain 5 of CD163 are resistant to porcine reproductive and respiratory syndrome virus 1 infection[J]. J Virol, 2018, 92(16): e00415-18.
[61] CHEN J, WANG H, BAI J, et al. Generation of pigs resistant to highly pathogenic-porcine reproductive and respiratory syndrome virus through gene editing of CD163[J]. Int J Biol Sci, 2019, 15(2): 481-492. doi: 10.7150/ijbs.25862
[62] YANG H, ZHANG J, ZHANG X, et al. CD163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus[J]. Antiviral Res, 2018, 151: 63-70. doi: 10.1016/j.antiviral.2018.01.004
[63] WELLS K D, BARDOT R, WHITWORTH K M, et al. Replacement of porcine CD163 scavenger receptor cysteine-rich domain 5 with a CD163-like homolog confers resistance of pigs to genotype 1 but not genotype 2 porcine reproductive and respiratory syndrome virus[J]. J Virol, 2017, 91(2): e01521-16.
[64] KARALYAN Z, ZAKARYAN H, ARAKELOVA E, et al. Evidence of hemolysis in pigs infected with highly virulent African swine fever virus[J]. Vet World, 2016, 9(12): 1413-1419. doi: 10.14202/vetworld.
[65] LILLICO S G, PROUDFOOT C, KING T J, et al. Mammalian interspecies substitution of immune modulatory alleles by genome editing[J]. Sci Rep, 2016, 6: 21645. doi: 10.1038/srep21645
[66] PAN D, LIU T, LEI T, et al. Progress in multiple genetically modified minipigs for xenotransplantation in China[J]. Xenotransplantation, 2019, 26(1): e12492.
[67] WANG Y, LEI T, WEI L, et al. Xenotransplantation in China: Present status[J]. Xenotransplantation, 2019, 26(1): e12490.
[68] 高孟雨, 杨光, 包骥. 利用CRISPR/Cas9技术繁育基因修饰猪在医学领域的研究进展[J]. 生物医学工程学杂志, 2018, 35(4): 637-642. [69] 黄耀强, 李国玲, 杨化强, 等. 基因编辑猪在生物医学研究中的应用[J]. 遗传, 2018, 40(8): 632-646. [70] BUTLER J R, WANG Z Y, MARTENS G R, et al. Modified glycan models of pig-to-human xenotransplantation do not enhance the human-anti-pig T cell response[J]. Transpl Immunol, 2016, 35: 47-51. doi: 10.1016/j.trim.2016.02.001
[71] ESTRADA J L, MARTENS G, LI P, et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/beta4GalNT2 genes[J]. Xenotransplantation, 2015, 22(3): 194-202. doi: 10.1111/xen.2015.22.issue-3
[72] 杨化强, 赖良学. 基因修饰猪作为人类疾病模型的研究进展[J]. 科技导报, 2011, 29(30): 57-62. doi: 10.3981/j.issn.1000-7857.2011.30.008 [73] 曾芳, 李紫聪, 董锐, 等. 转基因猪技术及其在农业上的应用[J]. 畜牧兽医学报, 2016, 47(2): 218-224. [74] 廖莎, 李国玲, 吴珍芳, 等. 转基因动植物生物反应器研究进展及应用现状[J]. 广东农业科学, 2018, 45(11): 126-136. [75] LEE M H, LIN Y S, TU C F, et al. Recombinant human factor IX produced from transgenic porcine milk[J]. Biomed Res Int, 2014, 2014: 315375.
[76] LU D, LIU S, SHANG S, et al. Production of transgenic-cloned pigs expressing large quantities of recombinant human lysozyme in milk[J]. PLoS One, 2015, 10(5): e123551.
[77] MA J, LI Q, LI Y, et al. Expression of recombinant human alpha-lactalbumin in milk of transgenic cloned pigs is sufficient to enhance intestinal growth and weight gain of suckling piglets[J]. Gene, 2016, 584(1): 7-16. doi: 10.1016/j.gene.2016.02.024
[78] PARK J K, LEE Y K, LEE P, et al. Recombinant human erythropoietin produced in milk of transgenic pigs[J]. J Biotechnol, 2006, 122(3): 362-371. doi: 10.1016/j.jbiotec.2005.11.021
[79] TONG J, WEI H, LIU X, et al. Production of recombinant human lysozyme in the milk of transgenic pigs[J]. Transgenic Res, 2011, 20(2): 417-419. doi: 10.1007/s11248-010-9409-2
[80] GORDON K, LEE E, VITALE J A, et al. Production of human tissue plasminogen activator in transgenic mouse milk[J]. Biotechnology, 1992, 24: 425-428.
[81] BLECK G T, WHITE B R, MILLER D J, et al. Production of bovine alpha-lactalbumin in the milk of transgenic pigs[J]. J Anim Sci, 1998, 76(12): 3072-3078. doi: 10.2527/1998.76123072x
[82] FANALI G, DI MASI A, TREZZA V, et al. Human serum albumin: From bench to bedside[J]. Mol Aspects Med, 2012, 33(3): 209-290. doi: 10.1016/j.mam.2011.12.002
[83] PENG J, WANG Y, JIANG J, et al. Production of human albumin in pigs through CRISPR/Cas9-mediated knockin of human cDNA into swine albumin locus in the zygotes[J]. Sci Rep, 2015, 5: 16705. doi: 10.1038/srep16705
[84] LI L, MENG H, ZOU Q, et al. Establishment of gene-edited pigs expressing human blood-coagulation factor VII and albumin for bioartificial liver use[J]. J Gastroenterol Hepatol, 2019.
[85] ZENG F, LI Z, ZHU Q, et al. Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors[J]. Sci Rep, 2017, 7: 41270. doi: 10.1038/srep41270
[86] HAI T, TENG F, GUO R, et al. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system[J]. Cell Res, 2014, 24(3): 372-375. doi: 10.1038/cr.2014.11
[87] ZHOU X, XIN J, FAN N, et al. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer[J]. Cell Mol Life Sci, 2015, 72(6): 1175-1184. doi: 10.1007/s00018-014-1744-7
[88] YANG D, YANG H, LI W, et al. Generation of PPAR gamma mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning[J]. Cell Res, 2011, 21(6): 979-982. doi: 10.1038/cr.2011.70
[89] CARLSON D F, FAHRENKRUG S C, HACKETT P B. Targeting DNA with fingers and TALENs[J]. Mol Ther Nucleic Acids, 2012, 1: e3.
[90] WANG Y, DU Y, SHEN B, et al. Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA[J]. Sci Rep, 2015, 5: 8256. doi: 10.1038/srep08256
[91] HUANG L, HUA Z, XIAO H, et al. CRISPR/Cas9-mediated ApoE-/- and LDLR-/- double gene knockout in pigs elevates serum LDL-C and TC levels[J]. Oncotarget, 2017, 8(23): 37751-37760.
[92] HOLM I E, ALSTRUP A K, LUO Y. Genetically modified pig models for neurodegenerative disorders[J]. J Pathol, 2016, 238(2): 267-287. doi: 10.1002/path.4654
[93] YANG H, WANG G, SUN H, et al. Species-dependent neuropathology in transgenic SOD1 pigs[J]. Cell Res, 2014, 24(4): 464-481. doi: 10.1038/cr.2014.25
[94] WANG G, YANG H, YAN S, et al. Cytoplasmic mislocalization of RNA splicing factors and aberrant neuronal gene splicing in TDP-43 transgenic pig brain[J]. Mol Neurodegener, 2015, 10: 42. doi: 10.1186/s13024-015-0036-5
[95] UCHIHARA Y, KATAOKA H, YOSHINO H, et al. Parkin mutation may be associated with serious akinesia in a patient with Parkinson's disease[J]. J Neurol Sci, 2017, 379: 119-121. doi: 10.1016/j.jns.2017.05.065
[96] VALENTE E M, SALVI S, IALONGO T, et al. PINK1 mutations are associated with sporadic early-onset parkinsonism[J]. Ann Neurol, 2004, 56(3): 336-341. doi: 10.1002/(ISSN)1531-8249
[97] ZHOU X, WANG L, Du Y, et al. Efficient generation of gene-modified pigs harboring precise orthologous human mutation via CRISPR/Cas9-induced homology-directed repair in zygotes[J]. Hum Mutat, 2016, 37(1): 110-118. doi: 10.1002/humu.22913
[98] WANG X, CAO C, HUANG J, et al. One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system[J]. Sci Rep, 2016, 6: 20620. doi: 10.1038/srep20620
[99] UCHIDA M, SHIMATSU Y, ONOE K, et al. Production of transgenic miniature pigs by pronuclear microinjection[J]. Transgenic Res, 2001, 10(6): 577-582. doi: 10.1023/A:1013059917280
[100] YANG D, WANG C E, ZHAO B, et al. Expression of Huntington’s disease protein results in apoptotic neurons in the brains of cloned transgenic pigs[J]. Hum Mol Genet, 2010, 19(20): 3983-3994. doi: 10.1093/hmg/ddq313
[101] BAXA M, HRUSKA-PLOCHAN M, JUHAS S, et al. A transgenic minipig model of Huntington’s disease[J]. J Huntingtons Dis, 2013, 2(1): 47-68.
[102] ROGERS C S, HAO Y, ROKHLINA T, et al. Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer[J]. J Clin Invest, 2008, 118(4): 1571-1577. doi: 10.1172/JCI34773
[103] ROGERS C S, STOLTZ D A, MEYERHOLZ D K, et al. Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs[J]. Science, 2008, 321(5897): 1837-1841. doi: 10.1126/science.1163600
[104] KLYMIUK N, BLUTKE A, GRAF A, et al. Dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle[J]. Hum Mol Genet, 2013, 22(21): 4368-4382. doi: 10.1093/hmg/ddt287
[105] YU H H, ZHAO H, QING Y B, et al. Porcine zygote injection with Cas9/sgRNA results in DMD-modified pig with muscle dystrophy[J]. Int J Mol Sci, 2016, 17(10).
[106] FLISIKOWSKA T, MERKL C, LANDMANN M, et al. A porcine model of familial adenomatous polyposis[J]. Gastroenterology, 2012, 143(5): 1173-1175. doi: 10.1053/j.gastro.2012.07.110
[107] SIKORSKA A, FLISIKOWSKA T, STACHOWIAK M, et al. Elevated expression of p53 in early colon polyps in a pig model of human familial adenomatous polyposis[J]. J Appl Genet, 2018, 59(4): 485-491. doi: 10.1007/s13353-018-0461-6
[108] ZHANG Y, XUE Y, CAO C, et al. Thyroid hormone regulates hematopoiesis via the TR-KLF9 axis[J]. Blood, 2017, 130(20): 2161-2170. doi: 10.1182/blood-2017-05-783043
[109] KANG J T, RYU J, CHO B, et al. Generation of RUNX3 knockout pigs using CRISPR/Cas9-mediated gene targeting[J]. Reprod Domest Anim, 2016, 51(6): 970-978. doi: 10.1111/rda.2016.51.issue-6
[110] GAO X, TAO Y, LAMAS V, et al. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents[J]. Nature, 2018, 553(7687): 217-221. doi: 10.1038/nature25164
[111] ROSS J W, FERNANDEZ D C J, ZHAO J, et al. Generation of an inbred miniature pig model of retinitis pigmentosa[J]. Invest Ophthalmol Vis Sci, 2012, 53(1): 501-507. doi: 10.1167/iovs.11-8784
[112] HE J, LI Q, FANG S, et al. PKD1 mono-allelic knockout is sufficient to trigger renal cystogenesis in a mini-pig model[J]. Int J Biol Sci, 2015, 11(4): 361-369. doi: 10.7150/ijbs.10858
[113] COOPER D K, SATYANANDA V, EKSER B, et al. Progress in pig-to-non-human primate transplantation models (1998-2013): A comprehensive review of the literature[J]. Xenotransplantation, 2014, 21(5): 397-419. doi: 10.1111/xen.2014.21.issue-5
[114] LAI L, KOLBER-SIMONDS D, PARK K W, et al. Production of α-1, 3-galactosyltransferase knockout pigs by nuclear transfer cloning[J]. Science, 2002, 295(5557): 1089-1092. doi: 10.1126/science.1068228
[115] DAI Y, VAUGHT T D, BOONE J, et al. Targeted disruption of the α1, 3-galactosyltransferase gene in cloned pigs[J]. Nat Biotechnol, 2002, 20(3): 251-255. doi: 10.1038/nbt0302-251
[116] HAUSCHILD J, PETERSEN B, SANTIAGO Y, et al. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases[J]. Proc Natl Acad Sci USA, 2011, 108(29): 12013-12017. doi: 10.1073/pnas.1106422108
[117] XIN J, YANG H, FAN N, et al. Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs[J]. PLoS One, 2013, 8(12): e84250. doi: 10.1371/journal.pone.0084250
[118] KANG J T, KWON D K, PARK A R, et al. Production of α1, 3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology[J]. J Vet Sci, 2016, 17(1): 89-96. doi: 10.4142/jvs.2016.17.1.89
[119] CHUANG C K, CHEN C H, HUANG C L, et al. Generation of GGTA1 mutant pigs by direct pronuclear microinjection of CRISPR/Cas9 plasmid vectors[J]. Anim Biotechnol, 2017, 28(3): 174-181. doi: 10.1080/10495398.2016.1246453
[120] LUTZ A J, LI P, ESTRADA J L, et al. Double knockout pigs deficient in N-glycolylneuraminic acid and galactose α-1, 3-galactose reduce the humoral barrier to xenotransplantation[J]. Xenotransplantation, 2013, 20(1): 27-35. doi: 10.1111/xen.2013.20.issue-1
[121] LI P, ESTRADA J L, BURLAK C, et al. Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection[J]. Xenotransplantation, 2015, 22(1): 20-31. doi: 10.1111/xen.2015.22.issue-1
[122] DENNER J. Why was PERV not transmitted during preclinical and clinical xenotransplantation trials and after inoculation of animals?[J]. Retrovirology, 2018, 15(1): 28. doi: 10.1186/s12977-018-0411-8
[123] 李晓开, 龙科任, 麦苗苗, 等. CRISPR-Cas9技术的原理及其在猪研究中的应用[J]. 生命科学, 2018, 30(6): 690-700. [124] YANG L, GUELL M, NIU D, et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs)[J]. Science, 2015, 350(6264): 1101-1104. doi: 10.1126/science.aad1191
[125] NIU D, WEI H J, LIN L, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9[J]. Science, 2017, 357(6357): 1303-1307. doi: 10.1126/science.aan4187
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