Base editing has the potential to improve important economic traits in agriculture and can precisely convert single nucleotides in DNA or RNA sequences into minimal double-strand DNA breaks (DSB). Adenine base editors (ABE) have recently emerged as a base editing tool for the conversion of targeted A:T to G:C, but have not yet been used in sheep. ABEmax is one of the latest versions of ABE, which consists of a catalytically-impaired nuclease and a laboratory-evolved DNA-adenosine deaminase. The Booroola fecundity (FecB B) mutation (g.A746G, p.Q249R) in the bone morphogenetic protein receptor 1B (BMPR1B) gene influences fecundity in many sheep breeds. In this study, by using ABEmax we successfully obtained lambs with defined point mutations that result in an amino acid substitution (p.Gln249Arg). The efficiency of the defined point mutations was 75% in newborn lambs, since six lambs were heterozygous at the FecB B mutation site (g.A746G, p.Q249R), and two lambs were wild-type. We did not detect off-target mutations in the eight edited lambs. Here, we report the validation of the first gene-edited sheep generated by ABE and highlight its potential to improve economically important traits in livestock.
oxide is involved in abscisic acid-induced photosynthesis and antioxidant system of tall fescue seedlings response to low-light stress, Environmental and Experimental Botany (2018),
A functional interpretation of filtered candidates and predicted regulatory pathways related to cashmere growth from sequencing trials needs available cell models, especially for hair matrix cells (HMCs), whose continual proliferation and differentiation result in rapid hair growth. To fulfill such goals, we herein obtained primary goat HMCs via a microdissection-based method; optimized the selection of the culture medium and coating substances for better cell maintenance; and exemplified their usefulness through examining the effects of calcium and all-trans retinoic acid (ATRA) on cells using immunoblotting, flow cytometry, and other techniques. As a result, we successfully acquired primary and passaged goat HMCs with typical keratinocyte morphology. Calcium-free RPMI (Roswell Park Memorial Institute) 1640 and MEM (minimum Eagle’s medium) outperformed normal DMEM/F12 (Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12) on long-term cell maintenance, whereas serum-free media K-SFM and EpiLife failed to support cell growth. HMCs differed molecularly and morphologically from their neighbor dermal papilla cells on expressions of feature genes, such as HOXC13, and on characteristic keratinocyte-like appearances versus fibroblast shapes, respectively. Higher calcium concentrations significantly stimulated the expression of the genes (e.g., KRT1 and IVL) involved in keratinocyte differentiation and, promoted cell proliferation. Moreover, 10−5 M ATRA obviously boosted goat HMC expansions and changed their cell cycle distributions compared to the controls. Our study shines a light on researches exploring the mechanisms underlying the growth of cashmere.
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