Construction of Lipopolysaccaride Binding Protein Knockout Mice Using CRISPR/Cas9 Technology

Abstract

ObjectiveTo construct a stable hereditary lipopolysaccaride binding protein (Lbp) gene knockout mice by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) gene editing technology.MethodsAccording to the sequence characteristics of Lbp gene in C57BL/6J mice, the target of sgRNA was designed, the 5'-end protein coding conserved sequence of Lbp gene was deleted and the shift mutation was introduced to inactivate LBP. The genome of F0, F1, F2, F3 generation mice was extracted; PCR was used to identify and sequence Lbp knockout; RT-PCR was used to verify Lbp gene transcription, and Western blotting was used to verify LBP protein expression in F2 generation.ResultsFive Lbp+/- mice from F0 generation, three Lbp+/- mice from F1 generation, four Lbp-/- mice from F2 generation and thirty Lbp-/- mice from F3 generation were obtained. RT-PCR showed that Lbp-/- mice mRNA was 244 bp and the translation was stopped early by code-shifting mutation. Western blotting showed that LBP protein was not expressed in the liver of Lbp-/- mice.ConclusionThe Lbp gene knockout mice were successfully constructed by CRISPR/Cas9 technique, which will provide a basis for further study of the immune and physiological effects of LBP.

Keywords

• lipopolysaccaride binding protein
• crispr / cas9 technology
• stable inheritance
• gene knockout mice