Generation of germ cell-deficient pigs by &lt;i&gt;NANOS3&lt;/i&gt; knockout

Kogasaka, Yuhei; Murakami, Sho; Yamashita, Shiro; Kimura, Daisuke; Furumoto, Yoshinori; Iguchi, Kana; Sendai, Yutaka

doi:10.1262/jrd.2022-028

Cited by 3 publications

(5 citation statements)

References 47 publications

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“…Ovaries from post-pubertal, NANOS3 KO, female mice and pigs exhibit diminished size and a lack of oogenesis compared to control, WT ovaries ( Tsuda et al, 2003 ; Suzuki et al, 2008 ; Kogasaka et al, 2022 ; Wang et al, 2023 ). Consistent with these findings, a previous bovine study reported that a NANOS3 KO female fetus at 190 days of fetal age displayed a complete absence of germ cells.…”

Section: Discussionmentioning

confidence: 96%

“…However, it is important to consider the differing developmental timepoints evaluated between these bovine studies. For instance, prior to puberty, NANOS3 KO pig ovaries were observed to have no discernible differences in appearance or size compared to their age-matched, control, WT counterparts ( Kogasaka et al, 2022 ; Wang et al, 2023 ). Yet, at the post-puberty stage (6 months old), the NANOS3 KO pig ovaries were noticeably smaller than those of WT pigs ( Kogasaka et al, 2022 ; Wang et al, 2023 ), which aligns with the findings observed in our current study in post-puberty bovines.…”

Section: Discussionmentioning

confidence: 99%

“…Nanos3 KO male mice have been reported to have atrophic testes, yet have intact seminiferous tubules, with no detectable spermatozoa ( Tsuda et al, 2003 ; Suzuki et al, 2008 ; Miura et al, 2021 ). Additionally, NANOS3 KO boars at 3 months old (pre-puberty) and at 6 months old (during puberty) also had no detectable germ cells, but had intact seminiferous tubules ( Kogasaka et al, 2022 ). In this study, the NANOS3 mosaic KO bull #838 was found to copy the phenotype of Nanos3 KO male mice and boars.…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, studies involving NANOS3 KO livestock have further demonstrated the conserved role of NANOS3 in germline development. Male and female NANOS3 KO pigs exhibited a complete loss of germ cells, while their gonadal development remained normal ( Kogasaka et al, 2022 ; Park et al, 2023 ; Wang et al, 2023 ). Similarly, in cattle, researchers produced a NANOS3 KO female fetus through somatic cell nuclear transfer (SCNT) cloning, which also showed a complete absence of germ cells but normal gonadal development ( Ideta et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

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Germline ablation achieved via CRISPR/Cas9 targeting of NANOS3 in bovine zygotes

Mueller,

McNabb,

Owen

et al. 2023

Front. Genome Ed.

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NANOS3 is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live NANOS3 knockout (KO) cattle have not been reported, and the specific role of NANOS3 in male cattle, or bulls, remains unexplored. This study generated NANOS3 KO cattle via cytoplasmic microinjection of the CRISPR/Cas9 system in vitro produced bovine zygotes and evaluated the effect of NANOS3 elimination on bovine germline development, from fetal development through reproductive age. The co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes (i.e., dual gRNA approach) at 6 h post fertilization achieved a high NANOS3 KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% (n = 8/26) pregnancy rate. A 75% (n = 6/8) total KO rate (i.e., 100% of alleles present contained complete loss-of-function mutations) was achieved with the dual gRNA editing approach. In NANOS3 KO fetal testes, PGCs were found to be completely eliminated by 41-day of fetal age. Importantly, despite the absence of germ cells, seminiferous tubule development was not impaired in NANOS3 KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, NANOS3 KO, germline-ablated bull was produced and at sexual maturity he exhibited normal libido, an anatomically normal reproductive tract, and intact somatic gonadal development and structure. Additionally, a live, NANOS3 KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in NANOS3 KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of NANOS3 KO cattle was unremarkable. Overall, this study demonstrated that the absence of NANOS3 in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of NANOS3 KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of NANOS3 function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in NANOS3 KO germline-ablated hosts.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Germline ablation achieved via CRISPR/Cas9 targeting of NANOS3 in bovine zygotes

Mueller,

McNabb,

Owen

et al. 2023

Front. Genome Ed.

View full text Add to dashboard Cite

show abstract

“…11 Histological findings revealed no germ cells in the testes or ovaries of Nanos3 mut/mut pigs recently created by chimeric embryo injections. 12 To improve upon chimeric embryo injections, which might yield mosaic pig models, the present study used somatic cell nuclear transfer-based cloning. The construction of non-mosaic, germline knockout Nanos3 À/À pigs with stable genotypes and phenotypes would be essential for germplasm preservation work.…”

mentioning

confidence: 99%