Generation of B6‐Ddx4^{em1(CreERT2)Utr}, a novel CreERT2 knock‐in line, for germ cell lineage by CRISPR/Cas9

Abstract

Germ cell development is essential for maintaining reproduction in animals. In postpubertal females, oogenesis is a highly complicated event for producing fertilizable oocytes. It starts when dormant primordial oocytes undergo activation to become growing oocytes. In postpubertal males, spermatogenesis is a differentiation process for producing sperm from spermatogonial stem cells. To obtain full understanding of the molecular mechanisms underlying germ cell development, the Cre/loxP system has been widely applied for conditional knock‐out mouse studies. In this study, we established a novel knock‐in mouse line, B6‐Ddx4 em1(CreERT2)Utr, which expresses CreERT2 recombinase under the control of the endogenous DEAD‐box helicase 4 (Ddx4) gene promoter. Ddx4 was specifically expressed in both female and male germ cell lineages. We mated the CreERT2 mice with R26GRR mice, expressing enhanced green fluorescent protein (EGFP) and tDsRed before and after Cre recombination. We found tDsRed signals in the testes and ovaries of tamoxifen‐treated B6‐Ddx4 em1(CreERT2)Utr::R26GRR mice, but not in untreated mice. Immunostaining of their ovaries clearly showed that Cre recombination occurred in all oocytes at every follicle stage. We also found 100% Cre recombination efficiency in male germ cells via the progeny test. In summary, our results indicate that B6‐Ddx4 em1(CreERT2)Utr is beneficial for studying female and male germ cell development.

Generation and characterization of a Ddx4‐iCre transgenic line for deletion in the germline beginning at genital ridge colonization

“…Conversely, Ddx4 iCre/iCre adult females were fertile (determined by breeding assessment; data not shown) and ovarian morphology appeared normal (Figure 2d). A male‐sterile phenotype was similarly observed in the tamoxifen‐inducible Ddx4 em1(CreERT2)Utr Cre allele, in which the transgene targeting design is highly similar to ours (Le et al, 2020). Interestingly, spermatogenesis in the Ddx4 ‐null allele is blocked in an earlier, premeiotic stage (Tanaka et al, 2000); presumably the hypomorphic nature of the Ddx4 iCreJoBo allele allows spermatogenesis to progress further through meiosis.…”

HDAC6-dependent deacetylation of NGF dictates its ubiquitination and maintains primordial follicle dormancy

“…We explored the potential function of HDAC6 during ovarian development and aging by first analyzing published transcriptome sequencing data from 3, 7, 14, 21, 60 dpp, 1-year, and 2-year mouse ovaries [55]. Specific expression of DDX4 and GDF9 were reported in the cytoplasm of oocytes [62,63], whereas NOBOX and Figlα were expressed in the nucleus of oocytes [16]. AMHR2 is the type II receptor for AMH, secreted by granulosa cells of small, growing follicles, and is an important clinical indicator of ovarian reserve [64].…”

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

“…Moreover, CreER T2 has been used extensively due to its robust activation at a specific stage upon TAM treatment with minimal background in Cre activity [34]. Given the unknown insertion site of Ddx4-CreER T2 acquired through random gene recombination, Hoai et al established a novel bicistronic mouse strain via CRISPR/Cas9, known as B6-Ddx4 em1(CreERT2)Utr [20]. However, infertility was found in the homozygous mouse line, manifesting as spermiogenesis arrest and absent mature spermatozoa.…”

“…The ATPase activity of Ddx4 was halved in homozygotes, possibly due to the additional 25 amino acid residues introduced by the P2A sequence, which protects Ddx4 from being knocked out. Additionally, Cre recombination is primarily activated in the testes and ovaries but not in the pancreas or thymus [20]. In summary, novel-inducible B6-Ddx4 em1(CreERT2)Utr mice are available for studying sterility at specific spermatogenic stages, and the use of the heterozygote Cre line during mating is recommended.…”

“…However, many subsequent reports with conditional ablations using these Cre have shown that they are not as specific or efficient as previously thought. Issues such as the efficiency and specificity of Cre recombinase not aligning with presuppositions need to be addressed given the widespread availability of abundant Cre-engineered lines, including heterotopic expression of non-target tissues [20,21], leakage [22], the instability of KO efficiency [17,23], and global KO [15,24]. Furthermore, various novel Cre mouse lines have been engineered with the request for more detailed dissection in critical cellular processes during spermatogenesis over the past decade (Figure 2).…”