An efficient i-GONAD method for creating and maintaining lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain.
An efficient i-GONAD method for creating and maintaining lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain.
复制标题
DOI：
10.1093/g3journal/jkab194
复制
发表时间：
2021-08-07
期刊：
影响因子：
--
通讯作者：
Iwamoto T
中科院分区：
文献类型：
--
作者：
Iwata S;Sasaki T;Nagahara M;Iwamoto T
由于有规则间隔的短回文重复序列/Cas系统的效率极高，纯合致死突变体的产生和维持往往是困难的。在这里，我们提出了一种高效的体内电穿孔方法，称为通过输卵管核酸传递（i-GONAD）改进的基因组编辑，其中致命基因中的两个等位基因中的一个在非靶向B6存在下被选择性编辑。从C3H/HeJJcl菌株中鉴定出C3H- in (6)1J倒置。该方法不需要对小鼠胚胎进行分离、培养、转移或其他体外处理。编辑后的致死基因在杂合子中稳定地保持着，因为在这个反转区间内重组被强烈抑制。利用这种策略，我们成功地产生了第一个具有胚胎致死突变的Tprkb零敲除菌株，并表明B6。C3H-In(6)1J能有效抑制重组。B6。C3H-In(6)1J被编码可见毛色标记Mitf的基因标记，Tprkb突变可以被视觉识别。我们列出了现有的平衡菌株，作为公共生物资源来制造这些致命的基因敲除。这种方法将允许更有效的实验，以进一步分析致命突变体。
As the efficiency of the clustered regularly interspaced short palindromic repeats/Cas system is extremely high, creation and maintenance of homozygous lethal mutants are often difficult. Here, we present an efficient in vivo electroporation method called improved genome editing via oviductal nucleic acid delivery (i-GONAD), wherein one of two alleles in the lethal gene was selectively edited in the presence of a non-targeted B6.C3H-In(6)1J inversion identified from the C3H/HeJJcl strain. This method did not require isolation, culture, transfer, or other in vitro handling of mouse embryos. The edited lethal genes were stably maintained in heterozygotes, as recombination is strongly suppressed within this inversion interval. Using this strategy, we successfully generated the first Tprkb null knockout strain with an embryonic lethal mutation and showed that B6.C3H-In(6)1J can efficiently suppress recombination. As B6.C3H-In(6)1J was tagged with a gene encoding the visible coat color marker, Mitf, the Tprkb mutation could be visually recognized. We listed the stock balancer strains currently available as public bioresources to create these lethal gene knockouts. This method will allow for more efficient experiments for further analysis of lethal mutants.
登录
查看更多内容
影响因子：
4.6
作者：
Takahashi G;Gurumurthy CB;Wada K;Miura H;Sato M;Ohtsuka M
通讯作者：
Ohtsuka M
影响因子：
9.5
作者：
Thorvaldsdóttir H;Robinson JT;Mesirov JP
通讯作者：
Mesirov JP
影响因子：
1.5
作者：
Nishijima, I;Mills, A;Bradley, A
通讯作者：
Bradley, A
影响因子：
64.8
作者：
Dickinson ME;Flenniken AM;Ji X;Teboul L;Wong MD;White JK;Meehan TF;Weninger WJ;Westerberg H;Adissu H;Baker CN;Bower L;Brown JM;Caddle LB;Chiani F;Clary D;Cleak J;Daly MJ;Denegre JM;Doe B;Dolan ME;Edie SM;Fuchs H;Gailus-Durner V;Galli A;Gambadoro A;Gallegos J;Guo S;Horner NR;Hsu CW;Johnson SJ;Kalaga S;Keith LC;Lanoue L;Lawson TN;Lek M;Mark M;Marschall S;Mason J;McElwee ML;Newbigging S;Nutter LM;Peterson KA;Ramirez-Solis R;Rowland DJ;Ryder E;Samocha KE;Seavitt JR;Selloum M;Szoke-Kovacs Z;Tamura M;Trainor AG;Tudose I;Wakana S;Warren J;Wendling O;West DB;Wong L;Yoshiki A;International Mouse Phenotyping Consortium;Jackson Laboratory;Infrastructure Nationale PHENOMIN, Institut Clinique de la Souris (ICS);Charles River Laboratories;MRC Harwell;Toronto Centre for Phenogenomics;Wellcome Trust Sanger Institute;RIKEN BioResource Center;MacArthur DG;Tocchini-Valentini GP;Gao X;Flicek P;Bradley A;Skarnes WC;Justice MJ;Parkinson HE;Moore M;Wells S;Braun RE;Svenson KL;de Angelis MH;Herault Y;Mohun T;Mallon AM;Henkelman RM;Brown SD;Adams DJ;Lloyd KC;McKerlie C;Beaudet AL;Bućan M;Murray SA
通讯作者：
Murray SA
影响因子：
30.8
作者：
Meehan TF;Conte N;West DB;Jacobsen JO;Mason J;Warren J;Chen CK;Tudose I;Relac M;Matthews P;Karp N;Santos L;Fiegel T;Ring N;Westerberg H;Greenaway S;Sneddon D;Morgan H;Codner GF;Stewart ME;Brown J;Horner N;International Mouse Phenotyping Consortium;Haendel M;Washington N;Mungall CJ;Reynolds CL;Gallegos J;Gailus-Durner V;Sorg T;Pavlovic G;Bower LR;Moore M;Morse I;Gao X;Tocchini-Valentini GP;Obata Y;Cho SY;Seong JK;Seavitt J;Beaudet AL;Dickinson ME;Herault Y;Wurst W;de Angelis MH;Lloyd KCK;Flenniken AM;Nutter LMJ;Newbigging S;McKerlie C;Justice MJ;Murray SA;Svenson KL;Braun RE;White JK;Bradley A;Flicek P;Wells S;Skarnes WC;Adams DJ;Parkinson H;Mallon AM;Brown SDM;Smedley D
通讯作者：
Smedley D