Gene Targeting

MGE core

  • Construct design expertise
  • Electroporation of ES cells; Selection; Cryoprotection; Expansion of clones for recombinant DAN analysis.
  • Expansion of recombinant ES cell clones
  • Blastocyst injection

PI laboratory

  • Construct generation
  • Development of genotyping assay
  • DNA preparation, Genotyping, Identification of recombinants
  • Southern Blot confirmation of recombinant stem cell clones

Recombinant ES cells - the fine print…

  • DNA: MGE will obtain at least 50µg of targeting construct DNA for electroporation, together with information on the structure of the construct, and the nature of the selectable marker genes present on the construct. Preferably, the targeting construct would be generated from genomic DNA of the mouse ES cell lines that are used by MGE. MGE will make such DNA available to Investigators. The Investigator will also provide proof of a valid genotyping assay for the targeted loci. The recommended method for genotyping is Southern Blot Hybridization of genomic DNA, although PCR methods are acceptable if multiple primer sets are used.
  • MGE will grow ES cell lines with proven germline transmission potential for genetic manipulation. MGE will document (i) plating efficiency and (ii) morphology of ES cells before each DNA transfer experiment, as well as (iii) plating efficiency and (iv) morphology of feeder fibroblast cells used in the experiment.
  • MGE will initially carry out two DNA transfer experiments by electroporation, using 25µg of construct DNA and a total of 5x107 ES cells.
  • MGE will perform the antibiotic selection of potentially recombinant ES cell clones, carry out the clonal expansion of individual ES cell clones, cryopreserve each clone twice (in plate form), and expand each clone for DNA preparation by the Investigator. MGE expects genotyping results in a timely fashion, typically within 2 months. Responsibility for genotyping lies solely with the Investigator.
  • The investigator will screen ES cell clones to determine which ES cell clones have undergone recombination. MGE will revive the identified clones from cryostorage, expand them, prepare another sample for cryostorage, and grow the cells up for confirmatory genotyping by the Investigator. Once confirmation is obtained that the ES cells in question carry the targeted allele, MGE will proceed to blastocyst injection of these ES cells.
  • MGE will initially generate 300 ES cell clones or 3 recombinant ES cell lines (as determined by genotyping in the Investigator's laboratory), whichever comes first. Should any of these targets not be met from the first ES cell/DNA transfer experiment, MGE will perform a second set of DNA electroporations into ES cells, and carry out the selection, expansion, and cryopreservation procedures. For the second experiment, MGE will obtain a new preparation of the DNA targeting construct from the Investigator. A third experiment can be considered, but only if one recombinant ES cell clone has been generated in the previous two experiments. Recovery of one recombinant clone demonstrates that the targeting construct is working, suggesting that a sufficient number of recombinants could be generated if more ES cell clones were generated and analyzed.
  • In case no recombinant ES cell clones can be recovered from two DNA transfer experiments, MGE will inform the Chair of the MGE Advisory Committee of it's efforts; at this stage it will be determined whether the structure or biological nature of the targeting construct may be the cause for a failure to create recombinant ES cells.

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