Resistance to blue ear disease and production performance assessment of CD163 gene-edited Large White pigs
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摘要:目的
运用CRISPR/Cas9基因编辑技术和体细胞核移植技术获得CD163基因全敲除(CD163 gene knockout，CD163-KO)的克隆猪，验证该基因敲除大白猪的抗蓝耳病特性，并研究其与野生型大白猪在生理、生产和繁殖性能等方面的差别，评估CD163-KO大白猪的主要生产性能。
方法用猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus，PRRSV)流行毒株NADC30-like对本研究所获得的11头CD163-KO大白猪和5头年龄、体质量相当的野生型大白猪进行攻毒试验。连续14 d观察猪的临床症状，记录直肠温度变化，检测血清中PRRSV病毒含量和抗体水平。14 d后取肺部组织进行切片，通过PRRSV免疫荧光试验观察肺脏感染情况。采集CD163-KO和野生型大白猪肺泡巨噬细胞进行免疫染色，观察肺泡巨噬细胞表面CD163蛋白的表达情况；采集猪外周血单核细胞进行诱导分化，观察CD163-KO与野生型大白猪诱导分化的巨噬细胞对血红蛋白−结合珠蛋白复合物的摄取情况。最后，统计分析CD163-KO与野生型大白猪的生产性能及公猪的繁殖性能。
结果本研究获得的CD163-KO大白猪对蓝耳病NADC30-like毒株具有完全抗性，敲除CD163基因不影响巨噬细胞的生理功能，CD163-KO与野生型大白猪的生长性能和繁殖性能无明显差异。
结论本研究是对CD163-KO猪抵抗蓝耳病的又一佐证和补充，证明CD163基因敲除操作对生产性能没有潜在的负面影响，为抗蓝耳病猪的生物安全提供了支撑。
Abstract:ObjectiveThe purpose of this study was to generate CD163 gene knockout (CD163-KO) Large White pigs by CRISPR/Cas9 gene editing and somatic cell nuclear transfer technologies, investigate the resistance to blue ear disease and the biosafety effect including physiology, productive and reproductive performances of the gene knockout pigs, and assess the main production performances of CD163-KO Large White pigs.
MethodIn this study, the 11 CD163-KO pigs and five age- and body weight-matched wild type Large White pigs were challenged with NADC30-like strain of porcine reproductive and respiratory syndrome virus (PRRSV). The rectal temperature, PRRSV antibody and virus variation were monitored continuously for 14 days. The lung tissues were examined by immunofluorescence of PRRSV antigen. Expression of CD163 protein on the surface of pulmonary alveolar macrophages in wild type and CD163-KO Large White pigs were examined through immunofluorescence staining. We compared the differentiation potential of monocytes into macrophages between CD163-KO and wild type pigs, and observed their uptake capacities to hemoglobin-haptoglobin complex. In addition, we analyzed the growth and reproductive production of the boars between CD163-KO pigs and wild type control to assess their biosafety and breeding value.
ResultCD163-KO pigs were completely resistant to NADC30-like strain without impairing the biological function associated with the modified gene, as well as productive and reproductive performances.
ConclusionThis study is an evidence and supplement of CD163-KO pigs resistance to blue ear disease, and demonstrates that CD163 gene knockout has no potentially negative effects on production performance, which provides evidences for the biosecurity of CD163-KO pigs.
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图 1 猪CD163基因示意图和CD163-gRNA序列
Figure 1. Genetic map of pig CD163 gene and sequence of CD163-gRNA
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图 2 野生型和CD163-KO大白猪取样组织中CD163蛋白表达
Figure 2. CD163 protein expression in sampling tissues of wild type and CD163-KO Large White pigs
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图 3 野生型和CD163-KO大白猪肺泡巨噬细胞表面CD163免疫荧光检测
Figure 3. Immunofluorescence detection of CD163 on the surface of pulmonary alveolar macrophages in wild type and CD163-KO Large White pigs
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图 4 野生型和CD163-KO大白猪攻毒后直肠温度、血清中PRRSV含量和抗体水平
Figure 4. Rectal pemperature, PRRSV load and antibody level in serum of wild type and CD163-KO Large White pigs post infection
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图 5 野生型和CD163-KO大白猪攻毒后第14天肺脏PRRSV免疫荧光结果
Figure 5. Immunofluorescence result of PRRSV in lung of wild type and CD163-KO Large White pigs at 14 d post infection
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图 6 野生型和CD163-KO大白猪巨噬细胞对血红蛋白−结合珠蛋白复合物的摄取能力
Figure 6. Uptake capacity of macrophages from wild type and CD163-KO Large White pigs to hemoglobin-haptoglobin complex
表 1 阳性单克隆细胞及CD163-KO大白猪测序结果
Table 1 Sequencing results of positive monoclonal cells and CD163-KO Large White pigs
类型 Type 靶位点序列(5′→3′) Sequence of target site 插入/缺失 Insertion/ Deletion 数量 Number 野生型大白猪 Wild type Large White pig GGTCGTGTTGAAGTACAACATGG 阳性单克隆细胞 Positive monoclonal cell GGTCGTGTTGAAGTACAAACATGG +A 3 CD163-KO大白猪 CD163-KO Large White pig GGTCGTGTTGAAGTACAAACATGG +A 18 
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表 2 野生型(n=10)和CD16-KO (n=5)大白猪生产性能比较1)
Table 2 Comparison of the productive performance of wild type and CD163-KO Large White pigs
项目 Item 野生型 Wild type CD163-KO P 出生体质量/kg Birth body weight 2.04±0.13 1.80±0.15 0.277 115 kg体长/cm Body length of 115 kg 119.8±1.2 119.2±1.0 0.746 115 kg体高/cm Body height of 115 kg 68.6±0.6 68.4±0.8 0.841 115 kg背膘厚/mm Backfat thickness of 115 kg 12.069±0.401 11.702±0.495 0.592 115 kg眼肌面积/cm2Eye muscle area of 115 kg 35.670±1.202 36.320±1.090 0.736 30 ~115 kg平均日增体质量/kg Average daily gain of body weight during 30−115 kg 1.075±0.047 1.008±0.068 0.428 30~115 kg饲料转化率 Feed/gain ratio during 30−115 kg 2.326±0.085 2.548±0.154 0.191 1)表中数据为平均值±标准误，P>0.05表示两组间差异不显著 (t检验) 1) Data are expressed as the mean ± standard error, P > 0.05 means there is no significant difference between two sets ( t test)
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表 3 野生型(n=20)与CD163-KO (n=10)大白猪精液质量比较1)
Table 3 Comparison of the quality of the semen from wild type and CD163-KO Large White pigs
项目 Item 野生型 Wild type CD163-KO P 精子总数/亿 Number of sperms 680.643 ± 84.535 685.726 ± 99.930 0.971 1∶1稀释后精子活力 Sperm motility after 1∶1 dilution 0.675 ± 0.013 0.700 ± 0.000 0.219 1∶1稀释后精子密度/(亿·mL−1) Sperm density after 1∶1 dilution 1.685 ± 0.195 1.578 ± 0.239 0.747 精子畸形率/% Abnormal rate of sperm 11.092 ± 1.358 10.000 ± 0.964 0.606 1)表中数据为平均值±标准误，P>0.05表示两组间没有显著差异(t检验) 1) Data are expressed as the mean ± standard error, P > 0.05 means there is no significant difference between two sets ( ttest)
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