Browsing by Author "Wells DN"

Now showing 1 - 4 of 4
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    Controlled Cytoplast Arrest and Morula Aggregation Enhance Development, Cryoresilience, and In Vivo Survival of Cloned Sheep Embryos
    (Mary Ann Liebert, Inc, 2021-02-02) McLean ZL; Appleby SJ; Fermin LM; Henderson HV; Wei J; Wells DN; Oback B
    Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphase-arrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.
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    Holstein Friesian dairy cattle edited for diluted coat color as a potential adaptation to climate change
    (BioMed Central Ltd, 2021-11-26) Laible G; Cole S-A; Brophy B; Wei J; Leath S; Jivanji S; Littlejohn MD; Wells DN
    BACKGROUND: High-producing Holstein Friesian dairy cattle have a characteristic black and white coat, often with large proportions of black. Compared to a light coat color, black absorbs more solar radiation which is a contributing factor to heat stress in cattle. To better adapt dairy cattle to rapidly warming climates, we aimed to lighten their coat color by genome editing. RESULTS: Using gRNA/Cas9-mediated editing, we introduced a three bp deletion in the pre-melanosomal protein 17 gene (PMEL) proposed as causative variant for the semi-dominant color dilution phenotype observed in Galloway and Highland cattle. Calves generated from cells with homozygous edits revealed a strong color dilution effect. Instead of the characteristic black and white markings of control calves generated from unedited cells, the edited calves displayed a novel grey and white coat pattern. CONCLUSION: This, for the first time, verified the causative nature of the PMEL mutation for diluting the black coat color in cattle. Although only one of the calves was healthy at birth and later succumbed to a naval infection, the study showed the feasibility of generating such edited animals with the possibility to dissect the effects of the introgressed edit and other interfering allelic variants that might exist in individual cattle and accurately determine the impact of only the three bp change.
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    Holstein Friesian dairy cattle edited for diluted coat color as adaptation to climate change
    (2020-09-17) Laible G; Cole S-A; Brophy B; Wei J; Leath S; Jivanji S; Littlejohn MD; Wells DN
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    Morula complementation restores male germline in NANOS2 null sheep
    (Oxford University Press on behalf of National Academy of Sciences, 2025-07-02) McLean ZL; Fermin LM; Appleby SJ; Wei J; Meng F; Maclean PH; Perry BJ; Brophy B; Turner P; Forrester-Gauntlett B; Wells DN; Snell RG; Oback B
    Current livestock breeding is slow to respond to rapidly mounting environmental pressures that threaten sustainable animal protein production. New approaches can accelerate genetic improvement by multiplying valuable embryonic, rather than adult genotypes. Chimeras, derived from complementing a sterile host with a fertile donor embryo, provide a pathway to multiply and exclusively transmit elite male germlines. We established genetically sterile hosts and optimized embryo complementation conditions to achieve absolute germline transmission in sheep. The spermatogonia-specific gene NANOS2 was disrupted in male (NANOS2+/−, NANOS2−/−) and female (NANOS2−/−) ovine fetal fibroblasts via gRNA–Cas9-mediated homology-directed repair. Targeted cell strains and wild-type controls were used to produce cloned offspring for breeding and phenotyping. Male homozygous knockout clones lacked detectable germ cells, while the somatic compartment of the testis remained intact. By contrast, male monoallelic and female biallelic targeting of NANOS2 did not affect germline development, resulting in fertile animals capable of producing fertile offspring with normal reproductive performance. The germ cell niche in NANOS2−/− hosts was most efficiently complemented by aggregating compacted morulae, rather than earlier cleavage stages, resulting in 97% blastocyst chimerization. Embryo-complemented cloned lambs from two different donor cell lines showed variable chimerism across tissues from each germ layer, including various degrees of germline colonization. A subset of germline chimeras contained normal numbers of prospermatogonia, indicating that the germline was fully restored for absolute transmission of the donor cell genotype.