Recent genome-wide studies have revealed that the majority of the mouse genome is transcribed as non-coding RNAs (ncRNAs) and growing evidence supports the importance of ncRNAs in regulating gene expression and epigenetic processes. However, the low efficiency of conventional gene targeting strategies has hindered the functional study of ncRNAs in vivo, particularly in generating large fragment deletions of long non-coding RNAs (lncRNAs) with multiple expression variants. The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has recently been applied as an efficient tool for engineering site-specific mutations of protein-coding genes in the genome. In this study, we explored the potential of using the CRISPR/Cas9 system to generate large genomic deletions of lncRNAs in mice. We developed an efficient one-step strategy to target the maternally expressed lncRNA, Rian, on chromosome 12 in mice. We showed that paired sgRNAs can precisely generate large deletions up to 23kb and the deletion efficiency can be further improved up to 33% by combining multiple sgRNAs. The deletion successfully abolished the expression of Rian from the maternally inherited allele, validating the biological relevance of the mutations in studying an imprinted locus. Mutation of Rian has differential effects on expression of nearby genes in different somatic tissues. Taken together, we have established a robust one-step method to engineer large deletions to knockout lncRNA genes with the CRISPR/Cas9 system. Our work will facilitate future functional studies of other lncRNAs in vivo.
Epididymis plays a crucial role in regulating the development of sperm motility and fertilizing capacity. To study the function of genes in the caput epididymidis using the Cre/loxP system, we generated Lcn5-Cre transgenic mice in which the expression of Cre recombinase is driven by the 1.8-kb Lcn5 promoter. A total of 11 founder mice carrying the Lcn5-Cre transgene were identified by PCR from 38 offspring, and the integration efficiency was 28.9%. However, only 1 of the 11 transgenic mouse lines were revealed with the Cre recombinase expressed specifically in caput epididymidis. Furthermore, expression of Cre mRNA was first observed on Postnatal Day 30 and continued to increase during development. Subsequently, Cre protein distribution was assessed by crossing Lcn5-Cre transgenic mice into the mT/mG reporter line. As expected, the Cre recombinase activity was only found in principal cells of the middle/distal caput epididymidis. The tissue-specific expression of Cre protein in the caput epididymidis was further confirmed using Lcn5-Cre mice crossed with a mouse strain carrying Aip1 conditional alleles (Aip1(flox/+)). In summary, a transgenic mouse line expressing Cre recombinase in principal cells of caput epididymidis was established. This transgenic mouse line can be used to generate conditional, caput epididymidis-specific knockout mouse models by crossing with mice harboring floxed (LoxP flanked) genes.
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