Tissue-Specific KO of ECM Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 168

8. It is extremely important to put each colony in a separated well. Keeping track of

the number of used yellow tips can help to avoid mistakes.

9. Obviously, it is essential to test that the floxed allele is fully functional before

starting the conditional deletion experiments.

10. Keep the glass ball clean. Wipe debris away with a piece of cloth. By accumulat-

ing glass debris, the ball eventually changes its size and subsequently the tem-
perature at which it can fuse with the needle.

11. Keep females well distributed in more than one cage. In this way, females will

not show a synchronized oestrus, thus enhancing the chance of finding individu-
als able to mate.

12. Making a few small air bubbles in the thinner part of the injection needle can

strongly increase the control over cell sucking and blowing.

13. Do not generate high pressure in the pipet. In this way transfer cannot be con-

trolled and often results in the loss of the embryos. If the pipet becomes clogged,
remove from the uterus and gently wash it in ES Medium, paying attention not to
lose the blastocysts.

14. Immediately after embryos are blown into the uterine cavity, the muscular wall

sometimes contracts and expels the transferred liquid together with injected blas-
tocysts. It is, therefore, useful to suck into the transfer pipet the liquid that tends
to overflow from the hole. In case embryos are expelled, they can be recovered in
the pipet.

15. Initially test DNA of a proven heterozygous animal in two different reaction tubes

each containing a separate combination of the two couples of oligonucleotides
(common, wild-type, or common, mutant allele). To simplify the procedure, test
whether all three oligonucleotides can work in one same reaction.

16. The success rate of this technique can vary depending on the nature of the tar-

geted locus and must therefore be empirically tested.

17. The GPI enzymes are homodimers and because dimerization occurs inside the

cell chimeric tissues show only two distinct bands. Three bands can be seen in
particular situations such as in skeletal muscle extracts. Myofibers containing
nuclei of distinct genotype can generate all three combination of subunits.

References
1. Evans, M. J. and Kaufman, M. H. (1981) Establishment in culture of pluripotent
cells from mouse embryos. Nature 292, 154-156.
2. Martin, G. (1981) Isolation of a pluripotent cell line from early mouse embryos
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USA 78, 7634-7638.
3. Smithies, O., Gergg, R. G., Boggs, S. S., Koralewski, M. A., and Kuckerlapati, M. S.
(1985) Insertion of DNA sequences into the human chromosomal β -globin locus

by homologous recombination. Nature 317, 230-234.
4. Thomas, K. R. and Capecchi, M. R. (1987) Site-directed mutagenesis by gene
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