3.3.1. Drug Selection
Positive selection of drug resistance markers whose coding sequences are incorporated into the targeting vector is used as the basis for selection in most gene targeting experiments. In the first instance, it is important to determine the sensitivity of newly isolated primary cultures to the chosen selection marker. Both the coding sequences for neomycin phosphotransferase (neo), which confers resistance to a neomycin analog, G418, and puromycin-N-acetlaytransferase (pac), which confers resistance to puromycin, have been used in targeting experiments in sheep.
1. Plate primary fibroblasts (5 x 104 total, approx 5% confluency), in duplicate, into the wells of a six-well plate.
2. After 24 h, add a range of G418 concentrations (0-600 |g/mL) or puromycin concentrations (0-1.5 |g/mL). Change the selection medium every 3-4 d for 10-12 d, at which stage killing of sensitive cells should be complete. The minimum concentration required for complete cell death is noted and subsequently used for selection of targeted cells (see Note 2).
3.3.2. Proliferation and Lifespan
A key parameter to the success of gene targeting in primary somatic cells is the proliferative vigour of the culture.
1. Use primary cultures with short doubling times in the order of 24 h or less because they speed up the timing of drug selection and expansion.
2. More important is the overall proliferative capacity of the culture to be used (see Note 3). Determine this as follows: seed duplicate 25-cm2 flasks with 2.5 x 105 cells. At subconfluence, trypsinize and count the cells. Seed a fresh flask with 2.5 x 105 cells (the aim is to achieve approx 1 in 10 split ratio). Calculate the number of population doublings (PDs; see Note 4). Continually repeat this process until the cells senesce.
This should be done immediately after the primary culture has been established. It should also be repeated with targeted colonies before they are considered for NT experiments.
1. Split confluent cultures of cells at a ratio of 1:2 into fresh flasks. Briefly trypsinise the cells and then pellet by centrifugation (200g for 5 min). Resuspend in a hypotonic solution of 0.4% trisodium citrate for 30 min to induce swelling.
2. Pellet by centrifugation (200g for 5 min). Resuspend by adding fixative (3:1 mix of methanol:acetic acid) dropwise while vortexing.
3. Repeat step 2 a further three times, before finally resuspending in 0.5 mL of the fixative (samples can be stored at -20°C at this stage).
4. Drop fixed cells on to cleaned (100% ethanol, 5% HCl solution) slides to ensure to an even spread of the chromosomes. After drying, stain slides with Gurr's Geimsa stain diluted 1:20 in phosphate-buffered water, pH 6.8 for 8 min. Chromosome number is routinely assessed from 30 spreads to ensure the normal chromosome complement (2n = 54 in sheep).
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