1. Is the w⁺ individual male or female? If the w⁺ is male and none of the offspring from the cross with yw are w⁺ males, then the insert resides on the X chromosome.
2. Which w⁺ G1 flies come from the same vial? Maybe designate them as 1A, 1B, 1C, etc. for one vial and 2A, 2B and so on for other vials. Those from the same vial are likely to have the insert in the same location since the transposition is an infrequent event. Those from separate vials cannot have the insert in the same location unless Go, transformed females mated with transformed siblings in the vial before each was mated individually to yw. Knowing if the Go fly was male or female can sort out whether this should be a concern.

1. Combine several of the G2 w⁺ males and females to maintain a stock. I will refer to this as the "interse" stock. After one more generation, the interse stock becomes a mix of homozygous and heterozygous flies. Sometimes, the homozygous flies have a more intense eye color than the heterozygous flies. By selecting the flies with the most intensely colored eyes each time you set up a new vial, the line may become homozygous. A rigorous test of this is to cross the putative homozygous flies with yw and see if all the offspring have colored eyes. We do not have enough experience with the white marker to judge how well interse approach works for generating homozygous stocks. Using virgins when you set-up new vials increases your chances of establishing a homozygous line. Note that if there are multiple inserts, you may have intensely colored eyes but be unable to establish homozygous lines.
2. Start the process of "homozygosing" and chromosome mapping.  Mate several G2 w⁺ males to several virgin yw; Sp/SM6a; Gl/TM3. This cross is the first step in using genetics to establish a homozygous line for either the 2^nd or 3^rd chromosome and for mapping which autosome carries the insert.
3. Prepare DNA from about 20 G2 flies and perform Southern blot analyses to definitively establish that the flies contain the expected insert. The Southern blot can also help determine the number of inserts.
4. Perform ß-galactosidase assays on heterozygous, colored-eyed flies.
5. Once lines are homozygous, analyses of the molecular architecture of the promoters can be undertaken.

For all of the inserts we have studied so far, analysis of EcoRV-cut DNA with a probe spanning the EcoRV site found in the ß-galactosidase gene will reveal the number of inserts in the gene. The DNA strider maps should be consulted to identify other digests that might provide a more diagnostic assessment of whether the correct insert is present. Ultimately, the inserts will be analyzed by genomic footprinting so the marker lane in these cases should be diagnostic of the insert.

One can determine quite early whether the promoter is active by taking a heterozygous or homozygous transformant and crossing it to yw. Colored-eyed adults derived from this cross must be heterozygous, and the level of promoter activity before and after heat shock can be determined by performing CPRG assays on extracts from the adult flies. White-eyed adults among the progeny can serve as negative controls. These measurements for mutants will be most meaningful when compared to a wild-type promoter construct.

A qualitative assessment of promoter activity can be obtained by staining tissues from heat shocked organisms with X-gal.  This X-gal staining procedure works best after the line is homozygous.  Otherwise, some of the homozygous larvae will lack the insert and there is not an easy way to distinguish these from larvae that carry the insert.

Within two months of first identifying a transformant, homozygous lines of flies should be established. At this time, it is appropriate to begin analyzing the chromatin structure of the promoters. Methods for genomic footprinting the promoters in salivary glands with permanganate and DNase I have been established in the lab. In addition, the more traditional method of probing the chromatin structure in nuclei from embryos with DNase I should be quite tractable if the flies lay lots of eggs. Some pilot experiments need to be done to determine how difficult it is to collect 0.5 to 1 gram of embryos and to determine if nuclei can be easily prepared. Early studies with the ry⁵⁰⁶ line were very tedious because many bottles of flies had to be set up in order to obtain enough eggs. The yw line is much more robust and this will hopefully be true of the transformants. I have a very effective protocol for 5 grams of embryos that should provide a good starting point for developing a scaled down protocol should it be easy to obtain embryos.