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Gene Function Analysis Using the Chicken B-Cell Line 209A thorough reading of Arakawa et al. (2) is highly recommended before attempting4-HT induction.d. Proceed with subcloning.4. Drug check after marker recycling or transfection: drug check is carried out forthree purposes: first, to ensure the excision of the marker cassette after markerrecycling, and second, to eliminate single targeted cells in a mix of targeted andnontargeted cells after the second transfection, and third, to confirm that the secondtransfection did not target the already knocked out locus thereby excising the firsttargeting event.a. Pick single colonies from the subcloning and transfer to a 96-well flat bottommicrotiter plate containing 300 µL CM/well.b. Transfer 75 µL of the cell suspension to serial wells containing 75 µL of thedrug(s) to be checked: blasticidin (60 µg/mL) or mycophenolic acid (2 µg/mL)or puromycin (2 µg/mL). End concentration is half that of the stock solution;30 µg/mL, 1 µg/mL, and 1 µg/mL, respectively.c. Let grow for 3–4 d at 41°C.d. Choose clones positive/negative for blasticidin, mycophenolic acid, or puromycinas expected.e. Cultivate selected clones for further experiments.5. This may facilitate the cloning of particularly difficult fragments. In using thisapproach, it is recommended to include the addition of a step to add the 3′A-overhang, as the long range PCR uses a proofreading enzyme.a. Method: 8 µL PCR product, 1 µL 10X PCR buffer, 0.1 µL dATP(10 µM), 0.1 µLTaq polymerase at 72°C for 10 min and then proceed according to the kitsinstructions. The target arms are then isolated from the prepared TA TOPO kitplasmid through RE as shown above.6. Drug check following second transfection.a. To check transfectants after the second transfection for the two drug-resistancemarker cassettes, the cells are cultivated in both drugs simultaneously.b. Mix 500 µL of each drug in a 24-well flat bottom microtiter plate and incubatewith 100 µL cell suspension.c. Let grow at 41°C for 3–5 d.7. Freezing positive clones:a. Positive clones are transferred to flasks and grown in 50 mL CM for 2 d.b. The cells are transferred to a 50-mL centrifuge tube and centrifuged for 5 minat 1500 rpm and 4°C.c. The supernatant is discarded and the pellet resolved in 10 mL freezing mediumcontaining DMSO.d. The cells should now be immediately transferred to 10 labeled cryovials andfrozen at –80°C. After 24 h (and up to 1 wk) the cells need to be transferred toa liquid nitrogen tank for long-term storage.e. Freezing medium: 70 mL (70%) CM, 20 mL (20%) FCS, and 10 mL (10%)DMSO.