Vps28基因敲除小鼠模型的构建及其对泌乳和免疫性状影响的研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0446

摘要/Abstract

摘要：

尽管基于QTL定位和关联分析研究挖掘了许多奶牛产奶性状候选功能基因,然而仅有少数几个基因经过体内外的功能验证。本课题组前期全基因组关联分析（GWAS）研究结果表明Vps28基因与奶牛产奶性状表型显著相关,并在奶牛乳腺组织中特异性高表达。为了深入了解该基因对泌乳性状的作用和功能,本研究利用CRISPR/Cas9技术构建了Vps28基因敲除小鼠,对其进行体内功能研究。研究结果显示Vps28基因敲除纯合型小鼠（Vps28-/-）极可能导致胚胎早期死亡,故只获得杂合型基因敲除小鼠（Vps28+/-）。敲除小鼠模型构建成功后,检测乳腺组织和脾脏组织中Vps28基因mRNA和蛋白的表达,结果显示与野生型小鼠（Vps28+/+）相比,Vps28+/-杂合型小鼠Vps28的mRNA和蛋白表达量均显著下降。进一步对母鼠的泌乳量、乳房形态和血常规进行检测,发现Vps28敲除母鼠的泌乳量下降,乳头明显凹陷,且血常规中白细胞数量、中性粒细胞数量、淋巴细胞数量和中间细胞数量显著低于野生型小鼠,推测Vps28基因不但影响泌乳,而且影响相关免疫性状。

关键词: CRISPR/Cas9, 基因敲除小鼠, Vps28基因, 泌乳, 免疫性状

Abstract:

Although many candidate genes for milk production traits have been mined based on QTL mapping and association analysis,only a few genes have been verified in vivo and in vitro. The results of our previous genome-wide association study（GWAS）showed that Vps28 was significantly associated with milk production traits in dairy cows. In addition,Vps28 was highly expressed in the mammary gland tissue of dairy cows. To further explore the role and function of Vps28 on milk production traits,CRISPR/Cas9 technology was used to construct Vps28 knockout mice,and its functions in vivo were studied. The results showed that Vps28 knockout homozygous mouse（Vps28-/-）were very likely to cause early embryonic lethality. Thus only heterozygous knock-out mice（Vps28+/-）were obtained in this study. Our results showed that the mRNA and protein expression in the mammary gland and spleen of Vps28 knockout mice（Vps28+/-）significantly decreased if compared with wild-type mice. Furthermore,Vps28 knockout mice had nipple dysplasia and reduced milk production. Also the white blood cells count,neutrophilic granulocyte count,lymphocyte count and intermediate cell count in the Vps28 knock-out mice were significantly lower than those in wild-type mice. These results suggest that Vps28 not only affects lactation,but also related immune traits in mice.

Key words: CRISPR/Cas9, knock-out mice, Vps28, lactation, immune traits

丁亚群, 丁宁, 谢深民, 黄梦娜, 张昱, 张勤, 姜力. Vps28基因敲除小鼠模型的构建及其对泌乳和免疫性状影响的研究[J]. 生物技术通报, 2022, 38(3): 164-172.

DING Ya-qun, DING Ning, XIE Shen-min, HUANG Meng-na, ZHANG Yu, ZHANG Qin, JIANG Li. Construction of Vps28 Knock-out Mice and Model Study of the Impact on Lactation and Immune Traits[J]. Biotechnology Bulletin, 2022, 38(3): 164-172.

图/表 11

图1 CRISPR/Cas9设计策略

Fig. 1 CRISPR / Cas9 design strategy

表1 sgRNA序列

Table 1 sgRNA sequence

sgRNA名称 sgRNA name	序列 Primer sequence（5'-3'）	PAM
5S2	GGCCATATTG GTAGGAGGTG AGG	AGG
3S2	GACCTACTCG GGCAGGACCC TGG	TGG

表2 引物序列

Table 2 Primer sequence

引物名称 Primer name	序列 Primer sequence（5'-3'）	产物长度 Product size/bp
3025-Vps28-5S-OuttF1	GGCTCTGCTTTCCAGGTCTCTTG	920
3025-Vps28-3S-OuttR2	GAGCAACTCACCACTGGCTGA- CTG
3025-Vps28-5S-intF1	GACCACACTGCTACGTCCATCC	521
3025-Vps28-5S-intR1	GTGACAGCCACATGGTGTTTGC

表3 qPCR引物序列

Table 3 Sequences of qPCR primers

引物名称Primer name	序列Primer sequence（5'-3'）
Vps28-F1	GCAGGATGTTCCACGGGATC
Vps28-R1	CTCCCGAGCATTCTTGTAGAGC
GAPDH-F1	GGTGCTGAGTATGTGGTGGA
GAPDH-R1	GGCATTGCTGACAATCTTGA

图2 Vps28敲除小鼠F0代基因型鉴定 B6为阴性对照,是B6基因组DNA;N为空白对照,无模板的对照;marker条带：2 000 bp,1 000 bp,750 bp,500 bp,250 bp,100 bp

Fig. 2 Genotype identification of F0 generation of Vps28-knockout mice B6 is negative control,which is B6 genomic DNA. N is blank control and control without template. Marker band：2 000 bp,1 000 bp,750 bp,500 bp,250 bp,and 100 bp

图3 Vps28敲除小鼠F0代基因型（-1 882 bp）测序结果

Fig. 3 Genotype sequencing results of F0 generation（-1 882 bp）of Vps28-knockout mice

图4 Vps28敲除小鼠F1代基因型鉴定 B6为阴性对照,是B6鼠基因组DNA;N为空白对照;Trans2K PlusII条带为：8 000 bp,5 000 bp,3 000 bp,2 000 bp,1 000 bp,750 bp,500 bp,250 bp,100 bp

Fig. 4 Genotype identification of Vps28-knockout mice in F1 generation B6 is negative control,which is the genomic DNA of B6 mice. N is blank control. The band of trans2K PlusII is：8 000 bp,5 000 bp,3 000 bp,2 000 bp,1 000 bp,750 bp,500 bp,250 bp,and 100 bp

图5 Vps28基因在脾脏和乳腺中的敲除效果 A：脾脏组织;B：乳腺组织

Fig. 5 mRNA and protein expresion of Vps28 in mouse spleen and mammary gland A：Spleen tissue. B：Mammary gland tissue. WT：wild type;KO：knockout. The same blelow

图6 母鼠后代仔鼠的体重及日增重

Fig.6 Average body weight and daily gain weight of offspring of WT and KT female rats

图7 Vps28基因敲除母鼠和野生型母鼠乳腺组织形态 A：野生型母鼠;B：Vps28基因敲除母鼠

Fig. 7 Structural morphology of mammary glands in WT-mice and Vps28-knockout mice A：WT mice. B：KO mice

图8 野生型小鼠和Vps28基因敲除小鼠血常规指标的比较

Fig. 8 Comparison of blood routine indexes between WT- mice and Vps28-knockout mice

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