Gene expression during development: Experimental design problem

Design experiments that would help you address the following questions. Begin by thinking about which basic techniques you already know and could use to help you answer each question. Then select the most appropriate experimental technique. (Remember: you are trying to design experiments to answer these questions; do not try to actually answer them right now.)

A. What is the first time point in early Drosophila larval development that the genes for Dorsal, its inhibitor, and the Toll receptor are all being expressed?

B. How could you compare total gene expression in larval versus adult Drosophila?

A. You could use PCR to address this question; to see if a gene is being expressed, we could look to see if the mRNA from that gene is present. A rough outline for the experiment might look like this: (1) Isolate RNA from larvae at several early time points in development (maybe 1 hour, 2 hours, and 3 hours after fertilization). We would do this at several time points in the hopes of finding the first time point when all three genes are being expressed. (2) Isolate the mRNA in these samples by using a poly-T "fishing" system. (3) Use reverse transcriptase to create cDNA (you could use a specific or general primer here). (4) Divide each sample up into three tubes, and run a different PCR reaction in each tube. Each reaction would use a different set of primers, one set for each gene we're interested in. (5) Look for PCR product in each tube by running a gel; if we can identify a time point when all three tubes contain PCR product, then we have answered our question.

Alternatively, you could use in situ hybridization to look for expression of these three specific genes in the larvae. In this case, you would create a probe for the mRNA from each of the three genes; you might choose to attach a different-colored fluorescent label to each probe so you could perform the in situ in one step. You would want each probe to be complementary to a region of the mRNA from each gene. Again, you would perform this experiment at several time points in early larval development, and look for the result which showed evidence for all three labels in the same larva.

Why would you not use a DNA microarray for this experiment? We are interested in relatively few genes in this instance, and it would not be cost effective to set up a microarray when you are only concerned about the expression of three genes. In addition, DNA microarrays are most useful for comparing two things (because two dye colors are typically used to label cDNA, see below); in this case we might need to compare results from several different developmental time-points.

B. This would be a good candidate for the use of a DNA microarray. Rather than comparing cDNA from two different organisms, you would compare cDNA from two distinct time points in development (in a larval stage and in an adult fly). To do this, you would isolate RNA from a larva and an adult fly, "fish" out the mRNA using a poly-T system, and use reverse transcriptase to create labeled cDNA (using a general, oligo-dT primer this time). Remember that we make labeled cDNA by including fluorescent nucleotides in our mix, and that we are not using dideoxynucleotides like we did in sequencing reactions. (Those ddNTPs were made without a 3' –OH group, so the growing nucleotide chains got terminated once a labeled ddNTP was added.) One of our samples (from the larva) would be labeled with one color fluorescent label (red or green), and the other sample (from the adult fly) would be labeled with the other color label. We would mix the cDNAs together, and incubate them (at room temperature, not at an elevated temperature) on a DNA microarray which contains single-stranded DNA from the coding strand of all the genes (exons only, no introns) from Drosophila. Remember that each dot on the microarray contains a different gene. We would wash the unbound cDNA off the microarray, and then view the microarray with a red laser and a green laser. One of our samples would produce green fluorescence if its cDNA bound to the spots, and the other sample would produce red fluorescence. Once our computer had interpreted the red results and the green results, it would produce one image with red, green, and yellow dots—the yellow color is the result of the computer finding both red and green signal on a spot. Yellow dots indicate that the gene is being expressed in both the larva and the adult.