Breeding and identification of Wuzhishan miniature pigs with α-1, 3 galactosyltransferase gene-knockout
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摘要:
目的 总结α-1，3半乳糖基转移酶（GGTA1）基因敲除（GTKO）五指山小型猪的繁育和鉴定。 方法 统计GTKO五指山小型猪的繁育结果及窝产仔数；采用PCR技术对GTKO五指山小型猪GGTA1基因敲除类型进行鉴定；采用荧光显微镜和流式细胞术对人、野生型五指山小型猪、GTKO五指山小型猪外周血单核细胞（PBMC）的αGal表型进行检测；比较GTKO五指山小型猪与野生型五指山小型猪血常规指标的差异。 结果 GGTA1基因型的遗传符合孟德尔定律；GGTA1－/－猪的PBMC流式检测无荧光表达，与基因型鉴定结果一致；GTKO五指山小型猪雌性猪初产窝产仔数为（6.8±1.8）只，经产窝产仔数为（8.3±2.2）只；血常规检测的各项指标与野生型五指山小型猪差异均无统计学意义（均为P＞0.05）。 结论 连续2代GTKO五指山小型猪遗传稳定，繁殖力正常。GTKO五指山小型猪可作为异种器官移植研究的可靠供体。 -
关键词:
- 琢-1, 3-半乳糖基转移酶 /
- 基因敲除 /
- 五指山小型猪 /
- 繁殖性能 /
- 琢-1, 3-半乳糖表型 /
- 外周血单
Abstract:Objective To summarize the breeding and identification of Wuzhishan miniature pig models with α-1, 3 galactosyltransferase (GGTA1) gene-knockout (GTKO). Methods The breeding and reproduction perform of GTKO Wuzhishan miniature pigs were assessed and the quantity of piglets was counted. The GTKO Wuzhishan miniature pig models with GGTA1gene knockout were validated by polymerase chain reaction (PCR). The αGal phenotype of peripheral blood mononuclear cells (PBMC) in human, wild-type Wuzhishan miniature pigs and GTKO Wuzhishan miniature pigs was detected by fluorescent microscope and flow cytometry. Routine blood test parameters were statistically compared between the GTKO and wild-type Wuzhishan miniature pigs. Results The inheritance of GGTA1 genotype complied with Mendel's law. Flow cytometry detected no fluorescent expression of PBMC in GGTA1－/－ pig models, which were consistent with the genotype identification results. The mean piglets of the primiparous GTKO Wuzhishan miniature pigs were (6.8±1.8) and (8.3±2.2) for the multiparous Wuzhishan miniature pigs. No statistical significance was noted in routine blood test parameters between the GTKO and wild-type Wuzhishan miniature pigs (all P>0.05). Conclusions Stable inheritance and normal reproductive capacity are observed in two generations of Wuzhishan miniature pigs continuously. GTKO Wuzhishan miniature pig is a reliable donor for heterogeneous organ transplantation. -
图 1 GTKO五指山小型猪繁育系谱图（部分）
○代表雌性猪；□代表雄性猪
Figure 1. Pedigree of GTKO Wuzhishan miniature pig (section)
图 2 荧光显微镜观察PBMC的αGal表型
Figure 2. αGal phenotype in PBMC detected by fluorescence microscopy
图 3 流式细胞术检测αGal表型
Blank为未染色五指山小型猪PBMC；Human为人PBMC；Control为野生型五指山小型猪PBMC；样品 (143、39-12、155) 为GTKO五指山小型猪PBMC
Figure 3. Flow cytometry analysis of αGal phenotype
表 1 本研究中使用的引物信息
Table 1. Information of primers used in the study
引物名称 引物序列（5’-3’） 引物用途 产物长度 退火温度（℃） TALEN-F1/R1 F1:AGGGACAGTAGACCTAGGAAAC 鉴定TALEN敲除类型 652 bp 60 R1: GATCCTAATTGGGTTTGCTGCC Neo-F/R F:GGATGCTTCCTCTAGTCTGTGATG 鉴定同源重组敲除类型 942 bp/ 2 005 bp 67 R:CTCTAGCCTACCCAGAACTGCAGAG 
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表 2 GTKO五指山猪产仔数量及存活仔数
Table 2. Litter amount and survival of piglets of GTKO Wuzhishan miniature pigs
代系 雌性猪 雄性猪 胎次（胎） 窝产仔数（只） 存活仔猪（只） F1 1（F0） 13 1 8 8 13 2 8 8 71 3 11 6 F2 71-3（F1） 71 1 4 3 71 2 10 7 71 3 10 9 1212（F1） 71 1 8 8 129 2 8 6 1316（F1） 129 1 6 6 129 2 5 4 32 3 6 6 21（F1） 71 1 8 7
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表 3 GTKO与野生型五指山小型猪血液生理指标的比较
Table 3. Comparison of blood routine indexes between GTKO and wild-type Wuzhishan miniature pig
血液生理指标 GTKO五指山小型猪（n=3） 野生型五指山小型猪（n=3） 五指山小型猪参考值[8] 白细胞计数 (109/L) 20.8±10.0 18.4±2.1 10.2~24.6 红细胞计数 (1012/L) 9.2±1.9 8.2±1.3 6.4~9.5 血红蛋白 (g/L) 166±37 177±62 142~217 红细胞比容（%） 45.6±9.4 50.1±18.4 38.4~69.2 红细胞平均容积（FL） 49.6±2.9 47.1±5.1 54.5~73.2 红细胞平均血红蛋白含量（pg） 18.0±0.9 16.7±1.5 16.5~22.5 红细胞平均血红蛋白浓度 (109/L) 363±9 355±7 299~329 红细胞分布宽度（%） 16.4±1.5 16.0±1.3 15.6~19.3 血小板计数（109/L） 409±316 345±61 144~387 血小板比容（%） 0.33±0.24 0.27±0.03 0.12~0.29 血小板平均容积（FL） 8.0±1.1 8.1±0.8 7.3~14.1 血小板分布宽度（%） 14.7±0.5 13.9±1.1 14.3~18.2 淋巴细胞计数 (109/L) 7.3±3.9 9.9±3.7 3.5~15.7 单核细胞计数 (109/L) 2.2±1.0 1.7±0.3 0.5~2.0 中性粒细胞计数 (109/L) 7.5±2.6 6.4±1.7 1.3~8.2 淋巴细胞百分数（%） 34.9±6.2 44.8±6.2 32.8~71.5 单核细胞百分数（%） 7.0±0.3 6.6±0.1 0.2~4.5 中性粒细胞百分数（%） 48.3±11.4 32.5±15.9 19.6~63.8
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