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

What is the benefit of integrating an extrachromosomal array? Extrachromosomal arrays suffer from three problems that may be solved to different extents by integration into the chromosomes:

1) Extrachromosomal arrays are lost from the germ line at some frequency, so maintaining a line carrying the array involves constantly selecting animals at least every few generations. For some phenotypes this is difficult. Integrating and homozygosing the array completely eliminates this problem. Having such a stable line is convenient for a variety of experiments. For example, when developing antibodies to a protein, it is useful to have overexpressing worms to stain, and to make extracts for westerns. You can use the overexpressor to work out conditions for using the antibody, and to confirm which band on the Western is really your protein.

2) Extrachromosomal arrays are lost mitotically at some frequency, so that animals that carry them are mosaics in which one cannot necessarily determine which cells have lost the array. This pattern of mosaicism varies in different individual worms. Thus transgenes on extrachromosomal arrays may not be expressed consistently in the set of cells that one would like. This problem is only partially solved by integrating the array. Results from integrating a number of beta-galactosidase reporter constructs show that genes in integrated arrays may be artifactually activated or shut off in certain cells. Presumably due to position effect, the integrated promoters may no longer give exactly the same expression pattern as the endogenous promoter in its normal chromosomal position. In addition, the pattern of expression from these integrated constructs is not 100% reproducible from animal to animal; some cells show variable expression. Overall, however, the expression from integrated arrays can be more reliable than that from extrachromosomal arrays. Basically, you're replacing the random error in expression that comes from mitotic loss of the extrachromosomal array with a more systematic error that comes from positional effects on the integrated array.

3) Extrachromosomal arrays can change their properties over time. The data for this is that if an extrachromosomal array has a certain measured transmission frequency, and a number of individual worms carrying this array are used to establish new lines, these new lines may have transmission frequencies for their arrays that strongly differ from each other and from the originally measured frequency. The expression of genes from these extrachromosomal arrays therefore potentially also suffers from this kind of variation. This creates the following kind of problem: if a transgene is to be crossed into a number of genetic backgrounds and the resulting phenotypes are to be compared, how can you know that the extrachromosomal array hasn't suffered some sort of change during the strain constructions? It is assumed (though with no data I'm aware of) that integrated transgenes are stable. Therefore, when you need your transgenes to have consistent properties over many generations, it is preferable to integrate them.

How to do it:

Summary: A strain bearing an extrachromosomal array is irradiated with gamma rays or x-rays, several hundred F1 progeny carrying the array are picked to individual plates, and for each F1 several F2 carrying the array are picked to individual plates. These plates are screened for 100% transmission of the array to the F3; such a strain is homozygous for an integrated array.

Stategy: Many people pick a few hundred F1s, and then pick 4-5 F2s from each F1. The large number of F2 plates involved usually necessitates doing the screen in a few batches to avoid killing yourself setting up all the F2s at once. However, both theoretically and practically, this is not the best strategy. Statistically, you will minimize the total number of plates set up (F1 and F2 summed) per integrant recovered if you pick more F1s, and pick only 2 F2s per F1. This also has the practical benefit of spreading the work out more evenly between the two generations. My practice is to pick 250 F1s on each of two consecutive days. After two days off, I then pick 2 F2s from each F1. For those rare F1 plates that seem to have thrown the array at a much higher frequency than the rest, I pick 4 F2s. After 1-2 days off, I score the F2 plates over three days. The hardest work is picking the F2's; I find that picking 500 on each of two consectutive days is about as much as is humanly possible to do.

1. Start by using microinjection to generate extrachromosomal arrays containing the gene of interest and an appropriate marker gene. In designing the experiment, consider using a coinjection marker like unc-76, dpy-20, or lin-15, rather than the popular dominant rol-6. With rol-6, the animals carrying the array are less healthy than the animals not carrying the array; with the other markers the reverse is true. I've found lin-15 to be a great marker; the Muv animals stick out like a sore thumb, making the screen for integrants a snap .