Browsing by Author "Wells DN"

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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.