ABCMdb

PMID: 10853269

Dickinson P, Kimber WL, Kilanowski FM, Webb S, Stevenson BJ, Porteous DJ, Dorin JR
Enhancing the efficiency of introducing precise mutations into the mouse genome by hit and run gene targeting.
Transgenic Res. 2000 Feb;9(1):55-66., [PubMed]

Sentences

No. Mutations Sentence Comment

12 ABCC7 p.Gly480Cys This new vector design allowed both efficient 'hit and run` for two cystic fibrosis (CF) mutations with no false positives and successful germline transmission of the novel G480C missense mutation. Login to comment 26 ABCC7 p.Gly480Cys Finally we designed a vector containing the base substitution that results in the missense mutation G480C. Login to comment 34 ABCC7 p.Gly480Cys Using these second generation vectors, ES cells could be 'hit` even more efficiently and ES cell 'run` clones bearing the precisely introduced G480C and F508 mutations were efficiently produced. Login to comment 35 ABCC7 p.Gly480Cys Chimaeric animals which have demonstrated their capability to transmit the G480C mutation through the germline have been generated. Login to comment 42 ABCC7 p.Gly480Cys The G480C mutation comprised three base substitutions at positions 1440, 1441, and 1443 (wild type sequence from position 1435 TCA GAG GGA ATT, mutant sequence from position 1435 TCA GAA TGC ATT). Login to comment 43 ABCC7 p.Gly480Cys The novel restriction sites SspI ( I507), Asp718I ( F508), and Nsil (G480C) were created along with the relevant mutations to allow screening for the presence of the mutation. Login to comment 120 ABCC7 p.Gly480Cys The grey star labelled 0.6 represents the novel 0.6-kb fragment generated by the introduction of the G480C linked Nsi site. Login to comment 173 ABCC7 p.Gly480Cys G480C After transfection of CGR8 cells with vector pHRG480C, 50 G418 resistant colonies were isolated and 38 analysed for targeting of the vector ('hit`) to the Cftr locus. Login to comment 175 ABCC7 p.Gly480Cys These clones were then screened to check for retention of the introduced mutation (a silent, novel restriction site introduced with the G480C mutation) during the targeting process. Login to comment 184 ABCC7 p.Gly480Cys These ES clones have subsequently been used for blastocyst injections to generate chimaeric mice which have demonstrated that they are capable of germline transmission of the G480C mutant allele. Login to comment 196 ABCC7 p.Gly480Cys A similar high frequency was observed both in the vector carrying the F508 mutation and the G480C mutation. Login to comment 198 ABCC7 p.Gly480Cys Detection and analysis of G480C 'hit and run` clones. Login to comment 202 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys (B) Analysis of DNA from G480C 'hit` clones to check for retention of the G480C mutation. Login to comment 203 ABCC7 p.Gly480Cys After digestion of DNA from 'hit` clones (6.2, 8.5, 3.3, 6.4, 9.3, 3.1, and 4.2) with NsiI and probing with the 1.3XH external probe the mutant 6.1-kb band indicates the presence of the G480C mutation at the Cftr locus in clones 6.2, 8.5, and 3.3 (see Figure 1(b) (iii)) for diagram). Login to comment 205 ABCC7 p.Gly480Cys (C) Verification that 'run` clones have retained the G480C mutation. Login to comment 207 ABCC7 p.Gly480Cys The 6.1-kb band indicates the presence of the G480C mutation (see Figure 1(b) (iii). Login to comment 242 ABCC7 p.Gly480Cys The fact that the 'run` clones selected in the F508 and G480C gene targeting experiments had not arisen by gene silencing rather than gene loss implied that the tk gene in these vectors was more resistant to inactivation than the fusion gene. Login to comment 245 ABCC7 p.Gly480Cys The F508 vector with this region of DNA still had a retention to loss ratio of 1:4 but analysis of the run clones with the G480C vector revealed that the ratio of mutation retention to loss was 1:1. Login to comment 246 ABCC7 p.Gly480Cys In conclusion, therefore, we demonstrate here that if careful vector design and culturing technique are used then the 'hit and run` strategy can be used efficiently to introduce predetermined mutations ( F508 and G480C) into murine ES cells. Login to comment 248 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys We have successfully used the ES cells carrying the G480C mutation to generate chimaeric mice which have gone on to demonstrate their ability to pass the G480C mutation through the germline. Login to comment