Construction of Vps28 Knock-out Mice and Model Study of the Impact on Lactation and Immune Traits

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

Abstract

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

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.

Figures/Tables 11

Fig. 1 CRISPR / Cas9 design strategy

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

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

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

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

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

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

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

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

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

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

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