Unlocking Genetic Mysteries during the Epic Sperm Journey toward Fertilization: Further Expanding Cre Mouse Lines

Abstract

The spatiotemporal expression patterns of genes are crucial for maintaining normal physiological functions in animals. Conditional gene knockout using the cyclization recombination enzyme (Cre)/locus of crossover of P1 (Cre/LoxP) strategy has been extensively employed for functional assays at specific tissue or developmental stages. This approach aids in uncovering the associations between phenotypes and gene regulation while minimizing interference among distinct tissues. Various Cre-engineered mouse models have been utilized in the male reproductive system, including Dppa3-MERCre for primordial germ cells, Ddx4-Cre and Stra8-Cre for spermatogonia, Prm1-Cre and Acrv1-iCre for haploid spermatids, Cyp17a1-iCre for the Leydig cell, Sox9-Cre for the Sertoli cell, and Lcn5/8/9-Cre for differentiated segments of the epididymis. Notably, the specificity and functioning stage of Cre recombinases vary, and the efficiency of recombination driven by Cre depends on endogenous promoters with different sequences as well as the constructed Cre vectors, even when controlled by an identical promoter. Cre mouse models generated via traditional recombination or CRISPR/Cas9 also exhibit distinct knockout properties. This review focuses on Cre-engineered mouse models applied to the male reproductive system, including Cre-targeting strategies, mouse model screening, and practical challenges encountered, particularly with novel mouse strains over the past decade. It aims to provide valuable references for studies conducted on the male reproductive system.

Keywords: Cre recombinase; Cre/LoxP; conditional knockout; epididymis; germ cells; testes.

Figure 1

(A) DNA excising mediated by the Cre-LoxP system. The mouse DNA was flanked with LoxP sites. Cre catalytic activity was driven by the exogenous promoters and removed the target exon between LoxP sites after Cre recombinase translocated into the nucleus. (B) Two kinds of gene recombination modes. (1) Gene integration via a conventional transgenic approach was inserted into the host genome randomly with the unpredictable recombination site and multiple integrations (left panel); (2) Cas9 was directed to the target locus and cut double-stranded DNA after binding to sgRNA. The exogenous genes were knocked-in by homology-directed repair efficiently and inherited stably (right panel).

Figure 2

Schematic diagram showing the established novel mouse strains and Cre lines used widely in the male reproductive system. (A) Cre models in the testes specific for each primary cell type, including PTM and peritubular myoid cells. SC, Sertoli cell; LC, Leydig cell; PGCs, primordial germ cells; Spg, spermatogonia; Spc, spermatocyte; Spd, spermatid; VE, vascular endothelial cells; MC, myeloid lineage cell (B) The Cre lines specific for IS, caput, corpus, cauda, and vas deferens.