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Gene Function Analysis Using the Chicken B-Cell Line 2012.2. Amplification of the Arms1. Chicken media (CM): add to 500 mL RPMI-1640 without glutamine (Gibco/BRL),50 mL (10%) FCS (Biochrom AG), 10 mL (2%) penicillin/streptomycin solution(Gibco/BRL), 5 mL (1%) glutamin solution (Gibco/BRL), 5 mL (1%) chicken serum(Sigma), and 0.05 mL (0.1%) of 1 M β-mercaptoethanol solution (Sigma) (see Note 1).2. Amplification reaction mixture (target arms PCR): 106.5 µL sterile H 2O, 15 µLcresol red, 15 µL buffer 1 (10X), 3 µL dNTP (10 mM), 1.5 µL polymerase mix,3 µL primer forward (25 pmol/µL), 3 µL primer reverse (25 pmol/µL), and 3 µLgenomic DNA (100 ng/µL). The mix is evenly divided among three PCR tubes.3. Buffer 1 and polymerase mix: Expand Long Template PCR System, (RocheApplied Science).4. Proteinase K buffer: 100 mM NaCl, 10mM Tris-HCL (pH 8.0), and 25 mM ethylenediaminetetra acetic acid (EDTA).5. TE (1X): 10 mM Tris-HCL (pH 8.0) and 25 mM EDTA.6. Expand Long Template PCR System (Roche Applied Science).7. Cresol red (Sigma-Aldrich).8. 10 mM dNTP (Fermentas).9. Standardized insert/vector RE reaction mixture: 30 µL PCR purification productor plasmid (~100 µg/µL), 4 µL appropriate buffer, 1 µL RE, 0.4 µL bovine serumalbumin (BSA), and 4.6 µL sterile H 2O.10. Buffer: use appropriate buffer supplied with the RE(s).11. Takara DNA Ligation Kit Ver. 2.1: 5 µL solution I, 0.5 µL prepared vector, and4.5 µL prepared fragment (Takara Bio Inc.).12. Chemically competent DH5α cells calcium chloride prepared.13. Amplification reaction mixture (colony PCR): 6.3 µL sterile H 2O, 1 µL dimethylsulfoxide (DMSO), 1 µL buffer S, 0.2 µL dNTP (10 mM), 0.7 µL Taq polymerase,2 µL primer forward (5 pmol/µL), 2 µL primer reverse (5 pmol/µL), and 10 µLpicked colony suspension.14. Buffer S: 166 mM (NH 4) 2SO 4, 670 mM Tris-HCl (pH8.8), 67 mM MgCl 2, and100 mM β-mercaptoethanol.15. Standardized RE analysis reaction mixture: 2.5 µL plasmid prep DNA, 14.3 µLsterile H 2O, 2 µL appropriate buffer, 0.2 µL BSA, 0.5 µL enzyme 1, and 0.5 µLenzyme 2 or sterile H 2O.16. Buffer: use appropriate buffer supplied with the RE(s).17. Linearization mixture: 300 µL Maxiprep DNA (1 µg/µL), 51 µL sterile H 2O,40 µL appropriate buffer, 4 µL BSA, and 5 µL NotI (or other enzyme).2.3. Knockout1. Blasticidin (stock solution 60 µg/mL) (Invitrogen).2. Mycophenolic acid (stock solution 2 µg/mL) (Sigma-Aldrich).3. Puromycin (stock solution 2 µg/mL) (Sigma-Aldrich).4. K buffer (10 µL): 0.5% Tween-20 and 100 µg/mL proteinase K in 1X PCR buffer(buffer 2 is used from the Expand Long Template PCR System [Roche AppliedScience]). Prepare just before use.