Services offered

a) Delivery of genomic ES DNA to test screening strategies and amplify isogenic ‘homology arms’

b) Advice on the design and construction of targeting vectors

c) Electroporation of the targeting vector into embryonic stem (ES) cells, selection and picking of double resistant clones.

d) Preparation of genomic ES cell DNA from selected clones in 96 well format and possibly a Southern blot from such DNA.

e) Culture of positive clones

f) Chimera production from positive clones by blastocyst injection or co-culture.

g) Delivery of chimeric mice to the collaboration partner.

Conditions requested

a) The targeting construct should be cloned by the user and supplied to the centre as a plasmid prep of not less than 200 µg in ‘Quiagen’-quality, together with a detailed restriction map and documented test digests

c) A Southern blot of ES cell DNA using an external probe (relative to the targeting construct) should be supplied to prove the detectability of the wild type allele by the proposed screening method.

d) If not done in the center, genomic DNA from positive ES cell clones should be tested using an external probe (by Southern blot or PCR) within 3 weeks of delivery. This is necessary because cryoconservation of ES cells in 96 well plates is not considered safe for long time storage and needs large freezer capacity.

e) An address to which chimeric mice (and/or offspring with germline transmission for the ES cells) can be shipped.