|
|
|
|
|
i-GONAD： improved genome-editing via oviductal nucleic acids delivery
i-GONAD：一种改进的经输卵管活体电转进行胚胎基因编辑的新技术
主讲人：Dr. Makoto Matsuyama, Shigei Medical Research Institute, Japan
仪器支持：NEPA21 高效基因电转系统
承办单位：华粤行仪器有限公司&NEPA GENE
时间、地点：4月1日（北京），4月3日（广州）
联系方式：18001171636；020-34821111 ext. 8245；cell@huayueco.com
|
仪器支持：NEPA21 高效基因电转系统
|
|
|
|
|
|
Dr. Makoto Matsuyama
松山博士是石井医学研究所PI，分子遗传学部门Division Head。他在国家基础生物学研究所获得博士学位，先后在发展生物学中心（CDB）和爱知（Aichi ）癌症研究中心工作。他的科学目标是利用遗传方法了解肾脏发育和病理学。他率先用NEPA21电转仪建立了i-GONAD法，成功地以相对较高的效率（eg. 敲除等位基因的效率约97%）生成了基因组编辑的小鼠；并应用于大鼠(rGONAD)，建立了Sprague-Dawley、 Lewis、 F1杂交（Sprague-Dawley和Brown-Norway）不同品系的KO\KI大鼠。i-GONAD技术将有望广泛用于其他哺乳动物的基因组编辑，为动物基因组编辑研究提供了新的思路和更简单、易行且高效的方法。
|
|
|
|
|
|
受精卵显微注射法或体外电穿孔法，是目前较常用的胚胎基因编辑获得KO/KI动物的方法。然而，这些技术需要费时费力的体外处理，包括受精卵分离、基因导入、体外培养、胚胎移植到代孕雌鼠等繁琐复杂流程。Dr. Makoto Matsuyama实验室开发了一种简单高效的体内电转方法，称为i-GONAD，改进的基因组编辑，通过输卵管核酸输送，不需要上述体外处理。目前i-GONAD法成功进行小鼠、大鼠基因组编辑，也有望应用于其他哺乳动物，如豚鼠、仓鼠、牛、猪、猴等。
|
|
|
rGONAD: Anewsimple in-vivo methodforgenome-editingin rats (KO\KI)
|
|
We termed this GONAD method as Rat improved GONAD (rGONAD).The rGONAD method involves two key steps: an injection with the solution containing Cas9 protein, guide RNA and single strand DNA (ssDNA) into the oviduct, followed by electroporation. Rats that have undergone rGONAD are bred as usual until birth, where offspring would be genome-edited and used for further analyses.
|
|
Moreover, i-GONAD is highly efficient in both knock-out and knock-in systems. We succeeded in generating genome-edited mice at relatively high efficiencies (for example, knockout alleles were produced at ~97% efficiency), and we found that i-GONAD can create genome-edited rats in various strains, including Sprague Dawley and Lewis, and F1 hybrids (between Sprague Dawley and Brown Norway), with efficiencies of ~62% for indel mutations and ~9% for knock-ins.
|
|
扫二维码，查阅文献
Successful production of genome-edited rats by therGONAD method
|
|
|
|
|
|
|
Day1
1.从孕鼠分离受精卵(显微操作)
2.体外受精卵显微注射或体外电转
3.体外培养受精卵至2细胞期
4.雌鼠与输精管结扎的雄鼠合笼交配
Day2
5.检查获得假孕雌鼠
6.将培养的胚胎移植到假孕雌鼠
|
|
|
|
|
|
1.注射：将cas9蛋白和gRNA\ssDNA注射入孕鼠输卵管
2.电转： NEPA21进行活体输卵管电转
|
|
|
|
★ 十几分钟即可完成，不需体外操作、培养、移植
★ 不需要代孕雌鼠及输精管结扎雄鼠
★ 孕鼠可重复使用，减少动物消耗
★ 不依赖熟练的胚胎操作人员
★ 适用于knock-out 和 knock-in
★ 更符合伦理学3Rs (Reduction, Replacement, and Refinement)要求
|
|
|
|
|
|
|
[1] Kobayashi T, Namba M, Koyano T, Fukushima M, Sato M, Ohtsuka M, Matsuyama M. Successful production of genome-edited rats by the rGONAD method.BMC Biotechnol. 2018 Apr 2;18(1):19
[2] Takabayashi S, Aoshima T, Kabashima K, Aoto K, Ohtsuka M, Sato M. i-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats.Sci Rep. 2018 Aug 13;8(1):12059.
[3] Yoshimatsu S, Okahara J, Sone T, Takeda Y, Nakamura M, Sasaki E, Kishi N, Shiozawa S, Okano H. Robust and efficient knock-in in embryonic stem cells and early-stage embryos of the common marmoset using the CRISPR-Cas9 system.Sci Rep. 2019 Feb 6;9(1):1528.
[4] Abbasi F, Miyata H1, Shimada K, Morohoshi A, Nozawa K, Matsumura T, Xu Z, Pratiwi P, Ikawa M. RSPH6A is required for sperm flagellum formation and male fertility in mice.J Cell Sci. 2018 Oct 11;131(19).
[5] Teixeira M, Py BF, Bosc C, Laubreton D, Moutin MJ, Marvel J, Flamant F, Markossian S. Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing.Sci Rep. 2018 Jan 11;8(1):474.
[6] Kaneko T, Sakuma T, Yamamoto T, Mashimo T. Simple knockout by electroporation of engineered endonucleases into intact rat embryos.Sci Rep. 2014 Oct 1;4:6382.
|
|
扫二维码，查阅
NEPA21 基因编辑其他文献
|
|
|
|
|
http://www.nepagene.jp/e_publications01.html#Embryos in oviduct
|
|
|
