CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and ...single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.
About 10% of male infertile patients show abnormalities in spermatogenesis. The microdeletion of azoospermia factor a (AZFa) region of the Y chromosome is thought to be a cause of spermatogenic ...failure. However, candidate gene responsible for the spermatogenic failure in AZFa deleted patients has not been elucidated yet. Using mice, we explored the function of Ddx3y, a strong candidate gene in the Azfa region, and Ddx3x, a Ddx3y paralog on the X chromosome, in spermatogenesis. We first generated Ddx3y KO male mice using CRISPR/Cas9 and found that the Ddx3y KO male mice show normal spermatogenesis, produce morphologically normal spermatozoa, and sire healthy offspring. Because Ddx3x KO males were embryonic lethal, we next generated chimeric mice, which contain Ddx3x and Ddx3y double KO (dKO) germ cells, and found that the dKO germ cells can differentiate into spermatozoa and transmit their mutant alleles to offspring by normal mating. We conclude that Ddx3x and Ddx3y are dispensable for spermatogenesis at least in mice. Unlike human, mice have an additional Ddx3y paralog D1pas1, that has been reported to be essential for spermatogenesis. These findings suggest that human and mouse DDX3 related proteins have distinct differences in their functions.
Meiosis is a hallmark event in germ cell development that accompanies sequential events executed by numerous molecules. Therefore, characterization of these factors is one of the best strategies to ...clarify the mechanism of meiosis. Here, we report tripartite motif-containing 41 (TRIM41), a ubiquitin ligase E3, as an essential factor for proper meiotic progression and fertility in male mice. Trim41 knockout (KO) spermatocytes exhibited synaptonemal complex protein 3 (SYCP3) overloading, especially on the X chromosome. Furthermore, mutant mice lacking the RING domain of TRIM41, required for the ubiquitin ligase E3 activity, phenocopied Trim41 KO mice. We then examined the behavior of mutant TRIM41 (ΔRING-TRIM41) and found that ΔRING-TRIM41 accumulated on the chromosome axes with overloaded SYCP3. This result suggested that TRIM41 exerts its function on the chromosome axes. Our study revealed that Trim41 is essential for preventing SYCP3 overloading, suggesting a TRIM41-mediated mechanism for regulating chromosome axis protein dynamics during male meiotic progression.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In mammals, sexual fate is determined by the chromosomes of the male and female gametes during fertilization. Males (XY) or females (XX) are produced when a sperm containing a Y or X-chromosome ...respectively fertilizes an X-chromosome-containing unfertilized egg. However, sexing of preimplantation stage embryos cannot be conducted visually. To address this, transgenic male mouse models with the ubiquitously expressed green fluorescent protein (GFP) transgene on X- (X-GFP) or Y-chromosomes (Y-GFP) have been established. However, when crossed with wild-type females, sexing of the preimplantation stage embryos by observing the GFP signal is problematic in some cases due to X-inactivation, loss of Y-chromosome (LOY), or loss of transgene fluorescence. In this study, a mouse model with the ubiquitously expressed red fluorescent protein (RFP) transgene on the Y-chromosome was generated since RFP is easily distinguishable from GFP signals. Unfortunately, the ubiquitously expressed tdTomato RFP transgene on the Y-chromosome (Y-RFP) mouse showed the lethal phenotype after birth. No lethal phenotypes were observed when the mitochondrial locating signal N-terminal of tdTomato (mtRFP) was included in the transgene construct. Almost half of the collected fertilized eggs from Y-mtRFP male mice crossed with wild-type females had an RFP signal at the preimplantation stage (E1.5). Therefore, XY eggs were recognized as RFP-positive embryos at the preimplantation stage. Furthermore, 100% sexing was observed at the preimplantation stage using the X-linked GFP/Y-linked RFP male mouse. The established Y-mtRFP mouse models may be used to study sex chromosome related research.
E26 avian leukemia oncogene 2, 3' domain (Ets2) has been implicated in various biological processes. An Ets2 mutant model (Ets2
), which lacks the DNA-binding domain, was previously reported to ...exhibit embryonic lethality caused by a trophoblast abnormality. This phenotype could be rescued by tetraploid complementation, resulting in pups with wavy hair and curly whiskers. Here, we generated new Ets2 mutant models with a frame-shift mutation in exon 8 using the CRISPR/Cas9 method. Homozygous mutants could not be obtained by natural mating as embryonic development stopped before E8.5, as previously reported. When we rescued them by tetraploid complementation, these mice did not exhibit wavy hair or curly whisker phenotypes. Our newly generated mice exhibited exon 8 skipping, which led to in-frame mutant mRNA expression in the skin and thymus but not in E7.5 Ets2
embryos. This exon 8-skipped Ets2 mRNA was translated into protein, suggesting that this Ets2 mutant protein complemented the Ets2 function in the skin. Our data implies that novel splicing variants incidentally generated after genome editing may complicate the phenotypic analysis but may also give insight into the new mechanisms related to biological gene functions.
•Cpne7Cre mouse is generated to analyze the function of Copine-7 and Cpne7+ neurons.•The theta oscillation during REM sleep is attenuated in Cpne7-KO mice.•REM sleep change in response to cage change ...or stress is altered in Cpne7-KO mice.•Cpne7+ neurons in the SLD regulate REM and NREM sleep.
Sleep is affected by the environment. In rodents, changes in the amount of rapid eye movement sleep (REMS) can precede those of other sleep/wake stages. The molecular mechanism underlying the dynamic regulation of REMS remains poorly understood. Here, we focused on the sublaterodorsal nucleus (SLD), located in the pontine tegmental area, which plays a crucial role in the regulation of REMS. We searched for genes selectively expressed in the SLD and identified copine-7 (Cpne7), whose involvement in sleep was totally unknown. We generated Cpne7-Cre knock-in mice, which enabled both the knockout (KO) of Cpne7 and the genetic labeling of Cpne7-expressing cells. While Cpne7-KO mice exhibited normal sleep under basal conditions, the amount of REMS in Cpne7-KO mice was larger compared to wildtype mice following cage change or water immersion and restraint stress, both of which are conditions that acutely reduce REMS. Thus, it was suggested that copine-7 is involved in negatively regulating REMS under certain conditions. In addition, chemogenetically activating Cpne7-expressing neurons in the SLD reduced the amount of REMS, suggesting that these neurons negatively regulate REMS. These results identify copine-7 and Cpne7-expressing neurons in the SLD as candidate molecular or neuronal components of the regulatory system that controls REMS.
Gene-knockout animal models with organ-deficient phenotypes used for blastocyst complementation are generally not viable. Animals need to be maintained as heterozygous mutants, and homozygous mutant ...embryos yield only one-fourth of all embryos. In this study, we generated organ-deficient embryos using the CRISPR-Cas9-sgRNA
system that induces cell death with a single-guide RNA (sgRNA
) targeting multiple sites in the genome. The Cas9-sgRNA
system interrupted cell proliferation and induced cell ablation in vitro. The mouse model had Cas9 driven by the Foxn1 promoter with a ubiquitous expression cassette of sgRNA
at the Rosa26 locus (Foxn1
; Rosa26_ms). It showed an athymic phenotype similar to that of nude mice but was not hairless. Eventually, a rat cell-derived thymus in an interspecies chimera was generated by blastocyst complementation of Foxn1
; Rosa26_ms mouse embryos with rat embryonic stem cells. Theoretically, a half of the total embryos has the Cas9-sgRNA
system because Rosa26_ms could be maintained as homozygous.
More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their ...essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories. Summary Sentence Thirty testis-enriched genes are dispensable for male fertility based on phenotypic analyses of knockout mice produced by the CRISPR/Cas9 system.
In mammals, more than 2000 genes are specifically or abundantly expressed in testis, but gene knockout studies revealed several are not individually essential for male fertility. Tesmin ...(Metallothionein-like 5; Mtl5) was originally reported as a testis-specific transcript that encodes a member of the cysteine-rich motif containing metallothionein family. Later studies showed that Tesmin has two splicing variants and both are specifically expressed in male and female germ cells. Herein, we clarified that the long (Tesmin-L) and short (Tesmin-S) transcript forms start expressing from spermatogonia and the spermatocyte stage, respectively, in testis. Furthermore, while Tesmin-deficient female mice are fertile, male mice are infertile due to arrested spermatogenesis at the pachytene stage. We were able to rescue the infertility with a Tesmin-L transgene, where we concluded that TESMIN-L is critical for meiotic completion in spermatogenesis and indispensable for male fertility. Summary sentence TESMIN protein, a member of metallothionein family, is essential for mouse spermatogenesis, especially in the meiotic stage.
The use of mice as experimental animal models has been a practice since the development of genetically engineered mouse models (GEMMs) in the early 1980s. New technologies, including genome editing, ...have helped in the time- and cost-efficient generation of GEMMs. However, methods for preparing pseudopregnant females, essential for the generation of GEMMs, remain less advanced. This study proposes a new method to achieve simple production of pseudopregnant female mice using a luteinizing hormone-releasing hormone agonist (LHRHa). A 20 µg LHRHa/mouse was identified as the best dose for inducing estrus synchronization. However, the frequency of copulation was 40% on a single injection. With sequential injections of 20 µg LHRHa/mouse on Days-1 and -2, followed by pairing on Day-5, 74% of LHRHa-treated females copulated with male mice. Moreover, LHRHa treatment did not affect fetal and postnatal development. Eventually, successful generation of offspring via embryo transfer was attained using LHRHa-treated pseudopregnant females. LHRHa administration method is efficient in producing pseudopregnant female mice for the generation of GEMMs, and we expect that it will contribute towards advancing the clinical research.
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