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210 Caldwell et al.8. Thawing cells:a. To thaw the cells, place the vial for 5 min in the 41°C incubator, then spindown the tube for 5 min at 1500 rpm and 4°C.b. Remove the freeze medium completely, resolve the pellet in 1 mL CM, andtransfer to a flask containing 25 mL CM.c. Let grow for 2–3 d at 41°C and check the cells condition every day under themicroscope.9. Subcloning by limited dilution:a. Count the viable cells using Trypan blue.b. Prepare three tubes containing 10 mL CM each and add 1000, 300, and 100cells, respectively.c. Plate each tube to a 96-well flat bottom microtiter plate by pipeting to each well100 µL (transfer three plates with 10 cells/well, 3 cells/well, and 1 cell/well).Alternatively, cells can be distributed across two 96-well flat bottom microtiterplates in a 300, 100, 30, 10, 3, and 1 cells/well configuration using a third of theplate for each dilution.d. Incubate the plates for 8 d without changing medium.e. Subclones should be visible by then as round colonies. Pick single coloniesinto 300 µL CM.References1. Buerstedde, J. M. and Takeda, S. (1991) Increased ratio of targeted to randomintegration after transfection of chicken B cell lines. Cell 67, 179–188.2. Arakawa, H., Lodygin, D., and Buerstedde, J. M. (2001) Mutant loxP vectors forselectable marker recycle and conditional knockouts. BMC Biotechnol. 1, 7.3. International Chicken Genome Sequencing Consortium (2004) Sequence andcomparative analysis of the chicken genome provide unique perspectives on vertebrateevolution. Nature 432, 695–716.4. International Chicken Polymorphism Map Consortium (2004) A genetic variationmap for chicken with 2.8 million single-nucleotide polymorphisms. Nature432(7018), 717–722.5. Schmid, M., Nanda, I., Hoehn, H., et al. (2005) Second report on chicken genesand chromosomes 2005. Cytogenet. Genome Res. 109(4), 415–479.6. Wahl, M. B., Caldwell, R. B., Kierzek, A. M., et al. (2004) Evaluation of the chickentranscriptome by SAGE of B cells and the DT40 cell line. BMC Genomics 5, 98.7. Caldwell, R. B., Kierzek, A. M., Arakawa, H., et al. (2005) Full-length cDNAsfrom chicken bursal lymphocytes to facilitate gene function analysis. GenomeBiol. 6, R6.8. Deng, C. and Capecchi, M. R. (1992) Reexamination of gene targeting frequencyas a function of the extent of homology between the targeting vector and the targetlocus. Mol. Cell Biol. 12(8), 3365–3371.