FGFR1基因敲除牛乳腺上皮细胞系的构建及功能研究

doi:10.16431/j.cnki.1671-7236.2026.01.029

Abstract

Abstract:

Objective This study aimed to establish a fibroblast growth factor receptor 1 （FGFR1） gene knockout bovine mammary epithelial cell line （bMECs） using CRISPR/Cas9 technology， and investigate the effects of FGFR1 gene deletion on cell viability， proliferation， and lactation function， thereby elucidating its molecular regulatory mechanisms in mammary gland development and lactation. Method sgRNAs were designed targeting exons 3 and 4 of FGFR1 gene. Following in vitro assessment of cleavage efficiency， a knockout plasmid for FGFR1 gene was generated. Genomic DNA was collected 7 d post-screening， and PCR amplification along with Sanger sequencing were employed to confirm FGFR1 gene editing. Monoclonal cells were isolated from the edited cell population using the limiting dilution method. FGFR1 gene knockout cell lines with a singular genotype were subsequently identified through TA cloning. The knockout efficiency of monoclonal cell lines was assessed via Western blotting， while cell viability was determined using CCK-8 assay. Additionally， Real-time quantitative PCR was utilized to evaluate the expression of genes associated with proliferation and lactation in the monoclonal cell lines. Result The in vitro cleavage assay demonstrated cleavage efficiencies of 82.87% and 27.80% for the two sgRNAs， confirming successful construction of the knockout plasmid. Subsequently， over 95% of bMECs displayed red fluorescence 72 h post-electrotransfection， with multiple double peaks observed in the target region following drug screening， indicating a pronounced editing effect. From a pool of 384 monoclonal cell lines， 92 were successfully propagated， with 72 （78.26%） exhibiting FGFR1 gene editing， 31 （33.70%） showing sgRNA1 site editing， and 55 （59.78%） displaying sgRNA2 site editing. Ultimately， 8 robust monoclonal cell lines were isolated. Sequencing analysis revealed diverse editing patterns among the monoclonal cell lines， including fragment deletions and base substitutions. Notably， the KO#2 cell line exhibited editing at two sites with a single editing type. Western blotting and CCK-8 results showed that compared with control group， the knockout effect of KO#2 strain cells was extremely significant （P<0.01）， while FGFR1 gene knockout did not significantly impact cell viability （P>0.05）. Real-time quantitative PCR analysis results revealed that compared with control group， there was no significant differences in the expression of genes associated with cell proliferation （P>0.05）. However， the significant reduction in the expression of genes related to lactation function was observed（P<0.05 or P<0.01）. Conclusion This study successfully constructed one strain of bMECs with FGFR1 gene knockout， indicate that the absence of FGFR1 gene did not notably impact cell survival and proliferation， but significantly inhibited lactation synthesis function. The results provided a basis for future investigations into the role and mechanisms of FGFR1 gene in bMECs.

Key words: FGFR1 gene; bovine mammary epithelial cell line （bMECs）; CRISPR/Cas9; gene editing; lactation regulation

CLC Number:

S823

GAO Linna, JIANG Yingying, HUANG Guangjun, WANG Yue, SHI Qianqian, WANG Huili, CHEN Kunlin. Construction and Functional Study of FGFR1 Gene Knockout Bovine Mammary Epithelial Cell Line[J]. China Animal Husbandry & Veterinary Medicine, 2026, 53(1): 317-332.

Figures/Tables 12

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引物 Primers	引物序列 Primer sequences （5′→3′）	片段长度 Product length/bp
QG-FGFR1-sgRNA1	F：CTTCGCCTGTCTTCCTTTGC	511
	R：GGACTTGGCTACCCACCTGA	511
QG-FGFR1-sgRNA2	F：AACCCCACCTATCCGTGAT	583
	R：GCTCGCCACCTGTAAACAAT	583

基因 Genes	登录号 Accession No.	引物序列 Primer sequences （5′→3′）	片段长度 Product length/bp
GAPDH	NM_001034034	F：GGCAAGTTCAACGGCACAGT	129
GAPDH	NM_001034034	R： CACCACATACTCAGCACCAGC	129
PCNA	NM_001034494	F： CGGACACGTTGGCACTAGTATT	138
PCNA	NM_001034494	R： CAGAAGGCATCTTTACTACACAGC	138
CyclinD1	NM_001046273	F： CCTGGTGAACAAACTCAAGTGG	140
CyclinD1	NM_001046273	R： CACAGAGGGCAACGAAGGT	140
CyclinE1	NM_001192776	F： CGGATACCATGAAAGAGGAAAG	162
CyclinE1	NM_001192776	R： CTCTATTACTGTCCCAAGGCTGA	162
CSN1S1	NM_181029	F： GCGTTACCTGGGTTATCTGGA	141
CSN1S1	NM_181029	R： ATCATAGGTTCTTTCTGTTGGGC	141
CSN2	XM_015471671	F： CAGCCTGAAGTAATGGGAGTCT	205
CSN2	XM_015471671	R： ACATGACAGTTGGAGGAAGAGG	205
CSN3	NM_174294	F： ACCAATACGCTGTGAGAAAGATG	156
CSN3	NM_174294	R： GGGTATGGCAGAAATTGATTATT	156
ELF5	NM_001024569	F： GAAGCCCTGGCGAAGATGT	118
ELF5	NM_001024569	R： TTCGGTCAACCCGTTCCA	118
GLUT1	NM_174602	F： CCTGGATGTCCTACCTGAGC	178
GLUT1	NM_174602	R： CGCACAGTTGCTCCACATAC	178
HKⅡ	XM_015473383	F： AAGATGCTGCCCACCTACG	123
HKⅡ	XM_015473383	R： TCGCTTCCCATTCCTCACA	123
SCD-1	NM_173959	F： CGACCTAAGAGCCGAGAAGC	162
SCD-1	NM_173959	R： CCAAGGGCATAACGGAATAA	162

Construction and Functional Study of FGFR1 Gene Knockout Bovine Mammary Epithelial Cell Line

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