title: Establishment of a mouse model to express bovine CD14 short hairpin RNA.
abstract: BACKGROUND
  Cluster of differentiation 14 (CD14) functions as a co-receptor for Toll-like receptor (TLR)-4 and myeloid differentiation factor (MD)-2 in detecting bacterial lipopolysaccharide. Together, these complexes promote the phagocytosis and digestion of Gram-negative bacteria, and initiate immune responses. To date, much of our understanding of CD14 function during Gram-negative bacterial inflammation comes from studies on mouse knockout models and cell transfection. To identify the effect of CD14 knockdown in this process in large livestock animals, we established a mouse model expressing bovine CD14 short hairpin (sh) RNA. shRNA fragments targeting bovine CD14 were screened by co-transfection in HEK 293 cells, and the most effective CD14 shRNA fragment was cloned into the eukaryotic expression vector pSilencer4.1-CD14 shRNA-IRES (internal ribosome entry site) and transferred into mouse zygotes by pronuclear microinjection to obtain transgenic mice. Expression of the enhanced green fluorescent protein (EGFP) reporter and genes related to the TLR4 signaling pathway was detected by immunohistochemistry (IHC) and quantitative polymerase chain reaction (PCR), respectively.
  
  
  RESULTS
  One effective shRNA fragment (shRNA-674) targeting bovine CD14 was obtained, the sequence of which was shown to be conserved between cows, buffalos, sheep, and humans. Thirty-seven founder pups were obtained by pronuclear microinjection, of which three were positive for the transgene. In the F(1) generation, 11 of 33 mice (33%) were positive for the transgene as detected by PCR. IHC analysis detected exogenous EGFP expression in the liver, kidney, and spleen of transgenic F(1) mice, indicating that they were chimeric. The expression of endogenous CD14 mRNA in the heart, liver, spleen, lung, and kidney of transgenic F(1) mice was decreased 8-, 3-, 19.5-, 6-, and 11-fold, respectively. The expression patterns of endogenous MD-2, TLR4, interleukin-6 and tumor necrosis factor-α genes in transgenic mice also varied.
  
  
  CONCLUSIONS
  This study confirms that transgenic mice expressing bovine CD14 shRNA can be generated by pronuclear microinjection, and demonstrates inhibited endogenous mouse CD14 expression that alters gene expression related to the TLR4 signaling pathway.
authors.affiliation: State Key Laboratory of Subtropical Bioresource Conservation and Utilization at Guangxi University, Nanning, Guangxi, China. xiangpingli@163.com.|Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, 530004, China. xiangpingli@163.com.
authors.forename: Xiangping
authors.initials: X
authors.lastname: Li
authors.affiliation: College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China. xiangp@gxu.edu.cn.
authors.forename: Shihai
authors.initials: S
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Subtropical Bioresource Conservation and Utilization at Guangxi University, Nanning, Guangxi, China. ypren@foxmail.com.|Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, 530004, China. ypren@foxmail.com.
authors.forename: Yanping
authors.initials: Y
authors.lastname: Ren
authors.affiliation: State Key Laboratory of Subtropical Bioresource Conservation and Utilization at Guangxi University, Nanning, Guangxi, China. mengail520@163.com.|Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, 530004, China. mengail520@163.com.
authors.forename: Meng
authors.initials: M
authors.lastname: Wang
authors.affiliation: College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China. kangchao8529@163.com.
authors.forename: Chao
authors.initials: C
authors.lastname: Kang
authors.affiliation: State Key Laboratory of Subtropical Bioresource Conservation and Utilization at Guangxi University, Nanning, Guangxi, China. 641241791@qq.com.|Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, 530004, China. 641241791@qq.com.
authors.forename: Liangliang
authors.initials: L
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Subtropical Bioresource Conservation and Utilization at Guangxi University, Nanning, Guangxi, China. ardsshi@163.com.|Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, 530004, China. ardsshi@163.com.
authors.forename: Deshun
authors.initials: D
authors.lastname: Shi
mesh_terms: D000818:Animals; D002417:Cattle / Q000235:genetics; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques / Q000662:veterinary; D057809:HEK293 Cells; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000235:genetics*; D008297:Male; D051379:Mice / Q000235:genetics; D008822:Mice, Transgenic / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D018950:Lipopolysaccharide Receptors; D034741:RNA, Small Interfering