Integrating extrachromasomal arrays into the C. elegans chromosomes原理及方法[Da

2. Selecting an extrachromosomal array to integrate:

When identifying transmitting lines, be sure to keep lines that transmit the array at a low frequency. About 20% transmission is very convenient; this is enough transmission to allow you to easily get enough animals to irradiate, but it is much lower than the 75% transmission frequency you will look for coming from animals heterozygous for an integrant. However, don't get bent out of shape looking for such a low transmitter, since this actually only makes a marginal (30%?) difference to the screen. Unfortunately, individuals from such lines tend to show huge variations in the rate at which they transmit their arrays, so that an animal that transmits the marker at a high frequency usually doesn't carry an integrated array. If all you have are high frequency transmitting lines, don't worry about it and forge ahead; you can still get the integrant by blindly picking F2s from each F1 and looking for an F2 that transmits the array to 100% of its progeny.

Another consideration is the strength the desired phenotype coming from your gene of interest in the array. You presumably should start with an extrachromosomal array that gives you a very strong phenotype. However, this is no guarantee that all the integrated lines generated from it will give a strong phenotype; position effects seem to introduce lots of variation, so that both strong and weak integrated lines will be produced.

3. You will mutagenize L4 hermaphrodites. In order to get enough L4 P0s carrying the array, you may need to set up lots of plates, especially if the array is transmitted at low frequency. For mutagenesis, use ~30 L4 animals (carrying the array) per large plate. You will need about 500 F1s (carrying the array) to get an integrant, so depending on the transmission frequency of your array and the number of integrants desired, you should scale up accordingly. (Data: using this screening strategy I got 3 integrants from 1500 F1s for one array tested).

When using lin-15 as a coinjection marker, you are faced with the annoying problem that lin-15 cannot be scored until the adult stage. Therefore, you should produce staged P0 animals to mutagenize by transferring lots of worms to a new lay plate twice a day. Then you can easily pick lots of L4 progeny to new plates to mutagenize. The day after mutagenesis, pick the non-Muv mutagenized animals to new plates. I put 30 on a small plate, and transfer them twice a day for 2 days to produce well staged F1 animals, so that non-Muv F1s can be easily picked as young adults.

4. Mutagenesis. Either x-rays or gamma rays seem to work; use 3600-4800 Rads with either type. The Horvitz lab has both an x-ray machine and a gamma source. Shai Shaham claims that either type of radiation works about the same. Yishi Jin says she gets consistent results with gamma rays, and that she got poor results the one time she used x-rays. I tried both x-rays and gamma rays on the same array and I got 2 integrants from 300 F1s with gamma rays, and 0 integrants from 200 F1s with x-rays.

5. When F1s can be scored for the presence of the array, pick the F1s to new plates (one F1 per plate). As mentioned above, you should pick about 500 F1s to get one integrant. . Most people avoid picking the very first F1 progeny that the P0s generate: these may be derived from germ cells that had already completed meiosis at the time of mutagenesis. Theoretically, (and with some empirical evidence), you can recover a higher frequency of mutants by waiting a bit. Typically the mutagenized animals are aged 24 hrs at 20deg., then transferred to new plates, and the F1s from this second set of plates are used.

7. When the F2 can be scored for the presence of the array, pick F2 animals to individual plates. As discussed above, I pick 2 F2s per F1.

If you are starting with an extrachromosomal array that is transmitted at a low frequency, you can also look through the plates for potential integrants. If you started with an array with a ~20% transmission frequency, you expect most of the background F1s to have thrown few animals with the array. F1 animals carrying a (heterozygous) integrated array, in contrast, will throw 75% F2s carrying the array. If you see a plate like this, pick a lot (~8) of F2s from it. Note that you will get some false positives and false negatives here. False positives: the array may still be extrachromosomal, but may have changed to a higher transmission frequency. False negatives: due to a position effect the marker on the array may be expressed only weakly, and may now be recessive instead of dominant. Another problem is that some arrays carry genes that make the worms sick. For example, the Rol phenotype makes worms grow slowly. Thus if the plates are scored too early, the percentage of animals carrying the array may be underestimated. One thing to do in this situation is to wait a few extra days to score the plates, and then look for the plates that are the last to starve out; these probably contained a high proportion of sick F2 worms (carrying the array).