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Poster Presentation : Gene Expression / Function

Production of FOXN1 Knockout Porcine Embryos Via Microinjection of CRISPR/Cpf1 mRNA

첫 페이지 보기
  • 발행기관
    한국동물생명공학회(구 한국동물번식학회) 바로가기
  • 간행물
    발생공학 국제심포지엄 및 학술대회 바로가기
  • 통권
    The 17th International Symposium on Developmental Biotechnology (2017.10)바로가기
  • 페이지
    pp.47-48
  • 저자
    Seon-Ung Hwang, Mirae Kim, Sang-Hwan Hyun
  • 언어
    영어(ENG)
  • URL
    https://www.earticle.net/Article/A347328

원문정보

초록

영어
Severe combined immune deficiency (SCID) pig is the important animal model for translational medical research, such as the development of humanized tissues and organs for transplantation and long-term evaluation of transplanted human cancer or stem cells. FOXN1 gene encodes a transcription factor essential for the development and function of thymic epithelial cells (TECs), the primary lymphoid organ that supports T-cell development and selection. This study was performed to produce the FOXN1 knockout (KO) SCID pigs’ embryos using the Crispr/Cpf1 system. Porcine genomic DNA sequences were analyzed, and the target sequences were selected using a web tool, Benchling (https://benchling.com/). The designed T7-crDNA oligos were synthesized by the Oligonucleotide Synthesis Service (Macrogen Inc., Seoul, Korea). pTE4396 (#74- 041; Addgene, Cambridge, MA, USA) vector was used as a template for AsCpf1 RNA synthesis. MEGAshortscript™ T7 Transcription Kit (Ambion, Austin, TX, USA) was used for the pFOXN1 target-crRNA synthesis, and RiboMAX™ Large Scale RNA Production Systems (Promega, Madison, WI) was used for the AsCpf1 RNA synthesis. The RNAs were diluted in microinjection buffer (10 mM Tris-HCl and 1 mM EDTA). 50 ng/μL of each pFOXN1 target-crRNA and 100 ng/μL AsCpf1 mRNA were co-injected into the cytoplasm of pronuclear stage PA embryos. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science) mean±SEM. As a result, the cleavage rates were no significant (p<0.05) difference between the control and microinjected groups (79.0±6.3 and 67.9±4.4), and there was no significant (p<0.05) difference in the development rate of blastocysts (60.6±12.1 and 49.3±6.6). These results suggest that there is no negative effect of embryo development by microinjection. To further analyze gene editing efficiency, it will be required to assay Indel and sequencing in the gDNA of a single blastocyst.

키워드

Porcine Crispr/Cpf1 FOXN1

저자

  • Seon-Ung Hwang [ Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Institute of Stem Cell & Regenerative Medicine, Chungbuk National University, Cheongju 28644, Republic of Korea ]
  • Mirae Kim [ Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Institute of Stem Cell & Regenerative Medicine, Chungbuk National University, Cheongju 28644, Republic of Korea ]
  • Sang-Hwan Hyun [ Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Institute of Stem Cell & Regenerative Medicine, Chungbuk National University, Cheongju 28644, Republic of Korea ]

참고문헌

자료제공 : 네이버학술정보

간행물 정보

발행기관

  • 발행기관명
    한국동물생명공학회(구 한국동물번식학회) [The Korean Society of Animal Reproduction and Biotechnology]
  • 설립연도
    1976
  • 분야
    농수해양>축산학
  • 소개
    동물번식생리학, 동물생명공학, 수의학, 인공수정 및 수정란이식을 이용한 동물개량에 관한 이론과 기술의 발전을 통해 학계, 연구계, 산업계 및 양축가 상호간의 협력을 도모함으로써 동물과학발전 및 사회일반의 이익에 기여 한다는 목적을 위해 노력해 나가겠습니다.

간행물

  • 간행물명
    발생공학 국제심포지엄 및 학술대회 [International Symposium on Developmental Biotechnology]
  • 간기
    연간
  • 수록기간
    2004~2018
  • 십진분류
    KDC 527 DDC 636

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