遗传修饰技术在绵羊分子设计育种中的应用

doi:10.16288/j.yczz.21-087

遗传 ›› 2021, Vol. 43 ›› Issue (6): 580-600.doi: 10.16288/j.yczz.21-087

遗传修饰技术在绵羊分子设计育种中的应用

王海涛^1,²(), 李亭亭³, 黄勋^1,², 马润林^1,^2,⁴(), 刘秋月¹()

1. 中国科学院种子创新研究院,北京 100101
2. 中国科学院遗传与发育生物学研究所,分子发育生物学国家重点实验室,北京 100101
3. 浙江农林大学,杭州 311300
4. 中国科学院大学,北京 100049

收稿日期:2021-03-08 修回日期:2021-04-14 出版日期:2021-06-20 发布日期:2021-04-30
通讯作者: 马润林,刘秋月 E-mail:wanght@genetics.ac.cn;rlma@ucas.ac.cn;qyliu@genetics.ac.cn
作者简介:王海涛,博士,博士后,研究方向：农业动物基因编辑。E-mail: wanght@genetics.ac.cn
基金资助:
中国科学院战略性先导科技专项(A类)资助编号(XDA24030205)

Application of genetic modification technologies in molecular design breeding of sheep

Haitao Wang^1,²(), Tingting Li³, Xun Huang^1,², Runlin Ma^1,^2,⁴(), Qiuyue Liu¹()

1. Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing 100101, China
2. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
3. Zhejiang A&F University, Hangzhou 311300, China;
4. University of Chinese Academy of Sciences, Beijing 100049, China

Received:2021-03-08 Revised:2021-04-14 Online:2021-06-20 Published:2021-04-30
Contact: Ma Runlin,Liu Qiuyue E-mail:wanght@genetics.ac.cn;rlma@ucas.ac.cn;qyliu@genetics.ac.cn
Supported by:
Supported by the Strategic Priority Research Program of Chinese Academy of Sciences No(XDA24030205)

摘要/Abstract

摘要：

利用遗传修饰技术可以使动物在遗传水平发生改变,实现在个体内表达外源基因或对其内源基因的功能造成影响。在动物育种中,可以利用遗传修饰技术在分子水平进行设计并实现品种的快速改良。从传统的遗传修饰技术、病毒载体、精子载体等介导的遗传修饰技术到新型人工核酸酶介导的基因编辑技术,尤其是CRISPR/Cas9为代表的人工核酸酶的运用使得基因编辑动物的制备变得更加高效快捷,并迅速在多个物种中得到应用。这些方法也已经拓展到了绵羊(Ovis aries)遗传育种领域。利用遗传修饰技术在绵羊中进行分子育种比传统的育种方式具有更大的优势,可以使用多种策略直接对性状进行快速改良,并且可以加快育种进程。本文详细介绍了遗传修饰技术在绵羊中的研究历程,探讨了通过遗传修饰技术进行绵羊分子设计育种的可能性,并提出以上技术和方法在绵羊育种中面临的问题和挑战。

关键词: 绵羊, 遗传修饰, 基因编辑, 动物育种

Abstract:

Genetic modification technologies can be used for modifying animal genome to express exogenous genes or affect the function of endogenous genes. In animal breeding, genetic modification technologies allow the rapid generation of germplasms with beneficial traits. It includes traditional genetic modification, virus or sperm carrier-mediated genetic modification and nuclease-mediated genome editing, especially the CRISPR/Cas9, one of the artificial nuclease genome editing technologies, have been applied in genome editing in many domestic animals including sheep (Ovis aries). Compared with conventional strategies used for animal breeding, there is great value for sheep breeding improvement by using genome editing technology, which is more effective and timesaving. In this review, we summarize the approaches of genetic modification in sheep and discuss the possibility of molecular design and breeding of sheep by genome editing technologies. We also identify the potential bottlenecks and challenges of these technologies in sheep breeding.

Key words: sheep, genetic modification, genome editing, animal breeding

王海涛, 李亭亭, 黄勋, 马润林, 刘秋月. 遗传修饰技术在绵羊分子设计育种中的应用[J]. 遗传, 2021, 43(6): 580-600.

Haitao Wang, Tingting Li, Xun Huang, Runlin Ma, Qiuyue Liu. Application of genetic modification technologies in molecular design breeding of sheep[J]. Hereditas(Beijing), 2021, 43(6): 580-600.

图/表 10

图1

图2

表1

表2

图3

表3

图4

表4

重要性状基因在基因修饰绵羊中的研究"

性状类型	基因名称	基因类型	基因功能	位点变化	基因修饰绵羊(预期)表型	参考文献
生长性状	GH	外源基因	促进生长	cDNA随机整合	生长加快	[6,18,19,21,22]
	MSTN	内源基因	抑制肌肉生长	RNAi敲低或敲除	敲除后肌肉量增加	[15,17,45, 48~50,56]
	SOCS2	内源基因	促进骨发育	单碱基突变	点突变体型增大,体重增加	[57]
羊毛纤维	CAT	外源基因	氯霉素抗性	cDNA随机整合	羊毛产量	[26]
	SLC7A11	内源基因	谷氨酸转移	cDNA随机整合	毛色变化	[34]
	FGF5	内源基因	调节毛发生长	定点敲除(移码突变)	敲除后毛产量增加	[38,45]
	ASIP	内源基因	黑皮质素拮抗剂	定点敲除(移码突变)	改变毛色	[58]
	IGF1	外源基因	生长调节	cDNA随机整合	外源基因表达后毛产量增加	[78]
抗病抗逆	vLRT/vvENV	外源基因	ENV蛋白	随机整合	产生抗体	[20]
	TLR4	外源基因	炎症反应	cDNA随机整合	增强抗病能力	[31,32]
	VP1 shRNA	外源基因	口蹄疫病毒蛋白	随机整合	干扰口蹄疫病毒基因组	[80]
繁殖性状	hα1AT	外源基因	蛋白酶抑制剂	Minigene随机整合	外源基因表达	[23]
	hFIX	外源基因	凝血	cDNA随机整合	外源基因表达	[24,25]
	HTT	外源基因	影响神经系统	cDNA随机整合	亨廷顿舞蹈症	[27,69]
	PPARγ	内源基因	脂肪沉积	cDNA随机整合	增加肌间脂肪	[35]
	LPL	内源基因	脂肪沉积	cDNA随机整合	增加肌间脂肪	[36]
	TNSALP	内源基因	降低碱性磷酸酶活性	单碱基突变	人HPP综合征模型	[52]
	PDX1	内源基因	胰腺发育	定点敲除(移码突变)	敲除后胰腺缺失	[53]
	CFTR	外源基因	膜信号转导调节	定点敲除(移码突变)	囊性纤维化模型	[55]
	BMPR1B	内源基因	卵泡发育	定点重组插入	增加产羔数	[59]
	otoferlin	外源基因	听力形成	定点重组插入	耳聋模型	[61]
	ASMT	外源基因	褪黑激素合成	cDNA随机整合	过表达增加褪黑激素表达量	[64]
基因表达	Rosa-26	友好位点	无	定点插入	外源基因插入后稳定表达	[54]

表4

Table 5

表6

在绵羊、山羊和猪中进行显微注射或体细胞核移植获得个体的比率统计"

物种	方法	基因	遗传修饰类型	移植胚胎数/ 受体数/怀孕	基因编辑后代/ 所有后代	获得后代比率(%)	参考文献
绵羊	原核注射	hGH	随机整合	1032/192/-	1/73	7.1	[6]
		hFⅨ	随机整合	67/24/11	6/7	10.4	[10]
		oGH	随机整合	408/109/39	4/44	10.7	[18]
		bGH; hGHRF	随机整合	842/98/33; 435/120/51	2/47; 9/63	5.6; 14.5	[21]
		bGH; hGRF	随机整合	247/-/42	11/42	17.0; 9.4	[22]
		hα1AT	随机整合	549/152/73	5/113	20.6	[23]
		hBCFVIII	随机整合	1145/454/276	13/434	37.9	[25]
		CAT	随机整合	371/76/-	4/31	8.4	[26]
		HTT	随机整合	413/138/-	6/150	36.3	[27]
		MSTN	Cas9敲除	213/55/31	2/35	16.4	[50]
		MSTN、ASIP和BCO2	Cas9敲除	578/82/77	35/36	7.3	[51]
		SOCS2	BE3点突变	53/8/3	3/4	7.5	[67]
		ASIP	Cas9敲除	92/60/6	5/6	6.5	[58]
		BMPR1B	Cas9点突变	279/39/16	7/21	7.5	[59]
		FGF5	基因敲除	100/53/14	3/18	18	[62]
		FGF5	基因敲除	63/43/18	20/23	36.5	[63]
	徒手克隆	fat-1	随机整合	53/29/4	3/3	5.7	[80]
	体细胞核移植	AAT	定点插入	80/42/20	13/14	17.5	[31]
		oTLR4	随机整合	859/113/12; 1009/108/7	9/9	0.7; 0.3	[32]
		fat-1	随机整合	128/16/3; 147/20/3	5/5	1.6; 2.0	[33]
		MSTN	TALEN敲除	70/5/1	1/1	1.4	[48]
		MSTN	TALEN敲除	282/37/28	13/23	1.4	[49]
山羊	原核注射	MSTN	Cas9敲除	416/137/64	26/93	22.4	[85]
		MSTN	Cas9敲除	18/5/3	1/4	22.2	[86]
		FGF5	BE3点突变	22/7/3	5/5	22.7	[87]
		BLG (β-lactoglobulin)	Cas9敲除	103/67/18	4	25.2	[88]
		GDF9	Cas9敲除	56/17/13	6/8	32.1	[89]
山羊	原核注射	MSTN	TALEN敲除	403/29/7	3/3	0.7	[90]
		MSTN	Cas9敲除	269/21/7	3/3	2.2	[91]
		EDAR (ectodysplasin receptor)	Cas9敲除	257/79/5	2/6	2.3	[92]
猪	体细胞核移植	-	纯体细胞克隆	586/10/2	0/7	1.2	[93]
		LDLR (low-density lipoprotein receptor)	敲除	2276/18/9	57/57	2.5	[94]
		PPARγ	ZFN敲除	1134/8/4	2/10	0.2	[95]
		GGTA1	TALEN敲除	2411/12/9	30/30	1.2	[96]
		FBXO40	Cas9敲除	5424/16/7	17/17	0.3	[97]
		CD163	Cas9敲除	2192/6/4	26/26	1.2	[98]
	显微注射	DMD (henne muscle dystrophy)	Cas9敲除	98/8/1	1/2	2.0	[99]
		TMPRSS2 (ransmembrane protease, serine S1, member 2)	Cas9敲除	135/2/2	12/12	8.8	[100]
		SRY (sex determining region Y)	Cas9敲除	66/4/2	12/12	18.2	[101]
		NANOS2	Cas9敲除	84/4/3	18/18	21.4	[102]
		GGTA1	Cas9敲除	94/4/2	4/15	16.0	[103]

表6

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遗传修饰技术在绵羊分子设计育种中的应用

Application of genetic modification technologies in molecular design breeding of sheep

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制备方法	基因名称	序列	编辑结果	表达情况	参考文献
显微注射	hGH	GH cDNA	随机整合	无	[6]
	oGH (ovine growth hormone)	GH cDNA	随机整合	表达	[18, 19]
	vLRT/vvENV (virus long terminal repeat/visna virus envelope)	病毒LTR区	随机整合	表达	[20]
	hGFR (human growth hormone-releasing factor)/ bGH (bovine growth hormone)	GH cDNA	随机整合	无	[21]
	hGFR/bGH	minigene	随机整合	表达	[22]
	hα1AT (human α1-antitrypsin)	minigene	随机整合	表达	[23]
	hAF IX (human antihemophilic factor IX)	cDNA	随机整合	表达	[24]
	hBCF VIII (human blood clotting factor VIII)	cDNA	随机整合	表达	[25]
	CAT (chloramphenicol acetyl transferase)	编码序列	随机整合	表达	[26]
	HTT (Huntington)	人HD cDNA	随机整合	表达	[27]
	IGF1 (insulin-like growth factors)	cDNA	随机整合	表达	[28]
	oTLR4 (ovine Toll-like receptor 4)	cDNA	随机整合	表达	[29]
体细胞核移植	hF IX (human factor IX)	完整编码区	随机整合	无	[10]
	oPRP和GGTA1	cDNA	定点敲除	GGTA1和PRP敲除	[30]
	COL1A1位点插入AAT基因	cDNA	定点插入	AAT表达	[31]
	oTLR4	cDNA	随机整合	表达	[32]
	Caenorhabditis elegans fat-1	cDNA	随机整合	被沉默	[33]

修饰方法	基因名称	辅助方法	编辑结果	参考文献
精子载体	SLC7A11	睾丸内注射	基因表达	[34]
	PPARγ	睾丸内注射	基因表达	[35]
	LPL	睾丸内注射	基因表达	[36]
	eGFP	慢病毒介导	基因表达	[46]
病毒载体	eGFP	显微注射	基因表达	[37~39]
	FGF5	显微注射	异位表达	[40]
	GFP	显微注射	基因表达	[41]
	tdTomato、zYellow和acGFP	显微注射	基因表达	[42]
转座子载体	Tn5和SB转座子	显微注射	GFP表达	[44]
转座子载体	SB转座子	RNAi	MSTN敲低	[45]

人工核酸酶	基因名称	递送方法	编辑结果	参考文献
ZFN	MSTN	脂质体转染	基因敲除	[14, 47]
TALEN	MSTN	显微注射	基因敲除	[15]
TALEN	MSTN	核移植	基因敲除	[48, 49]
CRISPR/Cas9	MSTN	显微注射	基因敲除	[17, 50]
	MSTN、ASIP (agouti gene)和BCO2 (beta-carotene oxygenase 2β)	显微注射	基因敲除	[51]
	TNSALP (tissue-nonspecific alkaline phosphatase)	显微注射	基因敲除	[52]
	PDX1 (pancreatic and duodenal homeobox protein 1)	显微注射	基因敲除	[53]
	GFP	显微注射	定点敲入	[54]
	CFTR (cystic fibrosis transmembrane conductance regulator)	核移植	基因敲除	[55]
	MSTN	核移植	基因敲除	[56]
	SOCS2 (suppressor of cytokine signaling 2)	显微注射	碱基突变	[57]
	ASIP	显微注射	基因敲除	[58]
	BMPR1B (bone morphogenetic protein receptor type 1B)	显微注射	基因敲除	[59, 60]
	otoferlin	显微注射	重组敲入	[61]
	FGF5	显微注射	基因敲除	[62, 63]
	ASMT (acetylserotonin methyltransferase)	显微注射	随机整合	[64]

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