Preparation of Infected Cells for Grafting

Transfected CEF cells expressing the gene of interest can be easily grafted into developing chick embryos (e.g., ref. 3). By using a host embryo from an infection-resistant strain, this technique also provides an effective method whereby infection can easily be limited to the implanted cells. Although it saves having to prepare high-titer viral stocks, infected cells must be cultured for each experiment.

1. To obtain enough cells for grafting, cells should be grown to confluence in a 250-mL tissue-culture flask. This can be achieved within 2 d after transferring cells from a confluent 10-cm tissue-culture dish.

2. Trypsinize cells and transfer to a sterile 15- or 50-mL plastic centrifuge tube. Spin at approx 3 K for 5 min. Remove supernatant, and resuspend cell pellet in 0.5 mL of CEF media.

3. Transfer the cells to a 1.5-mL Eppendorf tube, and pulse briefly (20 s) in a microfuge to pellet the cells.

4. Dislodge the cells carefully using a sterile tungsten needle, keeping the cell pellet as intact as possible (see Note 10).

5. Using a micropipet (see Note 11), transfer the cell pellet to a sterile 3.5-cm Petri dish containing 2 mL of CEF media. To avoid fragmenting the cell pellet, cut the end off the yellow or blue tip using sterile scissors. If the cell pellet does fragment while attempting to transfer it, respin and try again.

6. Place the Petri dish containing the cells in a 5% CO2 tissue-culture incubator at 37°C, and allow cells to consolidate for at least 1 h.

7. For grafting, dissect the cell pellet into suitable-size pieces under a dissecting microscope. Transfer the pellet to be grafted onto the embryo (see Note 12) using a micropipet, and position pellet appropriately using sterile tungsten needles (see Notes 13 and 14).

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