Transgenic F2 bovine embryos show stable germline transmission and maintenance of transgene expression through two generations

Gim, Gyeong-Min; Kwon, Dongil; Lee, Won-Wu; Jung, Dae-Jin; Kim, Dae Hyun; Yi, Junkoo; Jang, Goo

doi:10.1093/biolre/ioaa165

Cited by 3 publications

(1 citation statement)

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“…The SV40 promoter has been found to maintain more steady levels of expression when stably integrated in mammalian cells as compared to the CMV promoter [31]. A recent paper reported transgenic cattle expressing GFP driven by the human elongation factor 1α promoter showed stable GFP expression over 6 years and F2 germline transmission without gene silencing [32]. It is also possible that the presence of multiple copies of the transgene in the complex alleles in the current study may also lead to repeat-induced gene silencing.…”

Section: Discussionmentioning

confidence: 99%

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes

et al. 2021

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Background The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells. Results By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template. Conclusion The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

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Section: Discussionmentioning

confidence: 99%

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes

et al. 2021

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Long-term (10-year) monitoring of transposon-mediated transgenic cattle

Yum,

Choi,

Gim

et al. 2024

Transgenic Res

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The production of transgenic animals using non-viral methods has raised questions regarding their long-term health and genomic stability. In this study, we evaluated these aspects in transgenic cattle over ten years, using transposon-mediated gene transfer. Our longitudinal analysis included a comprehensive health assessment and whole-genome DNA resequencing. We found no significant alterations in physiological parameters or health complications in transposon-mediated transgenic cattle that exceeded 10 years of age. Genomic analysis revealed that the rates of somatic mutations and copy number variations in transgenic cattle were comparable to those in non-transgenic cattle. Furthermore, structural variants were infrequent, suggesting that transposon-mediated gene insertion did not compromise genomic integrity. These findings highlight the viability of transposon systems for generating transgenic livestock, potentially expanding their applications in agriculture and biotechnology. This study contributes significantly to our understanding of the long-term implications of transgenesis in large animals and supports the safety and stability of this method.

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