Biopolym. Cell. 2019; 35(6): 427-436.
Molecular and Cell Biotechnologies
Generation of transgenic ducks by crispr/CAS9-mediated gene inser-tion combined with the sperm-mediated gene transfer (SMGT)
1, 3Konoval O., 5Korol P., 1Tabaka P., 4Kostenko S., 1Lizhi L., 4Chepiha A., 4Doroshenko M., 4Drahulian M., 1Xingchen B., 2Xuetao H., 2Liumeng L.
  1. Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences
    198 Shiqiao Road, 310021, Hangzhou, Zhejiang, China;
  2. Zhuji Guowey Poultry Development Co, Ltd, Ltd.
    1 Gujing Road, 311813, Wangjiajing, Zhuji, Zhejiang, China;
  3. Ukrainian Laboratory of Quality and Safety of Agricultural Products of National University of Life and Environmental Sciences of Ukraine,
    Mashynobudivnykiv str., 7, 08162, Chabany, Kyiv region, Ukraine;
  4. National University of Life and Environmental Sciences of Ukraine,
    Heroiv Oborony St., 15, 03041 Kyiv, Ukraine;
  5. Institute of Animal Breeding and Genetics nd. a. M.V. Zubets of NAAS of Ukraine,
    Pohrebniaka Str. 1, 083213, Chubyns’ke, Kyivs’ka obl., Ukraine

Abstract

Aim. To edit the duck genome by the methods of HDR-directed integration of the EGFP gene into the duck host genome in combination with SMGT using CRISPR / Cas9. Methods. The implementation of the HDR-mediated gene of green fluorescent protein (EGFP) was achieved by the combined action of four plasmids. The first pX330 plasmid contained the Cas9 gene. Two other plasmids contained sgRNA genes: pBR322-sgRNA1 and pBR322-sgRNA2. The fourth pBR322-HDR-EGFP plasmid was constructed to contain the DNA vector with left homologous sequence part(LHP), the EGFP gene coding sequences and the right homologous sequence part(RHP). The DNA sequence data for designing HDR-EGFP-insert and guide 20-nucleotide long parts of sgRNA 1 and sgRNA 2 were taken from the genome DNA sequense [sequence] data of «Anas platyrhynchos Spindlin 1 (SPIN1) gene. There were 24 ducks (13 males and 11 females) of the Shaoxing breed used for this experiment. The sperm transfection was performed using reagent Lipofectamine 2000. Results. As a result of these experiments 31 ducks were obtained, 19 of which carried the (F2) analysis revealed that in total 16 ducks (F1) (14 females and 2 males) transmitted the transgene DNA to their offsprings. The results showed that 27.6 % (56/203) of F2 descendants were positive for the transgene DNA construct. Conclusions. The results indicate that exogenous DNA was successfully inserted into the duck genome.
Keywords: CRISPR/Cas9, EGFP, SMGT, Transgenic poultry, Duck

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