You may have been lucky enough to have spent Valentine’s Day with a loved one last week, but were you a successful match maker like me? I was Cupid for the day on the 14th of February; introducing specially selected females, to their perfect male matches. Not only did I set up these couples, but I also get to look after their children that were conceived since that memorable first encounter. This may seem exciting, but I should probably add that this was done for scientific purposes, and the couples involved were Drosophila melanogaster, commonly known as fruit flies. Nonetheless I would love to tell you all about my work, as the cupid of the fly lab.
The hidden agenda behind my matchmaking was to mate two different fly lines and create offspring with a desired genotype. Drosophila have four pairs of chromosomes, with half coming from the father and the other half coming from the mother. This allows us to create offspring with an expected pair of chromosomes by crossing parents with known genotypes. In my case the female flies, numbered 411548-411558, carry a micro RNA (miRNA) gene that is under the control of an upstream activating sequence (UAS) on the third chromosome. Strains 41149, 41152 and 41155 have the miRNA balanced over Sb1, which is a gene that causes the bristles on the back of the flies to be stubbly instead of long as found in the wildtype form. The 5535 males carry a GAL4 gene under the control of a promoter called ‘Eyeless’ on the second chromosome and are balanced over CyO. ‘Eyeless’ is a homologue of human Pax6 and is needed for normal neurodevelopment of the brain, particularly in the eyes. CyO is a gene that causes the wings of the flies to be curly instead of the straight wildtype form. Figure 1 shows the genes on chromosomes 2 and 3 in the female and males in an example cross of a 5535 male and 41149 female.
Figure 1: Schematic of chromosomes 2 and 3 in the 5535 male and 41149 female. Where chromosomes 1 and 4 are both wild type.
The outcome of these crosses is expected to be flies with heterogeneous chromosome 2 (GAL4 with the Eyeless promoter and wild type) and heterogeneous chromosome 3 (miRNA gene with the UAS promoter and wild type), see Figure 2 for more details. UAS only promotes the miRNA expression if GAL4 is present, therefore, the parents will not express miRNA but the progeny will. The way in which we ensure the offspring has all the desired genes, will be discussed in a future post.
Figure 2: Schematic of desired progeny genes on chromosome 2 and 3. GAL4 is expressed and binds UAS which allows for the miRNA encoded on chromosome 3 to be expressed.
Let’s not worry about the children for now, instead let’s concentrate on the matchmaking! The first blind date for these flies wasn’t far off a candle light dinner in a fancy restaurant as they met each other in a vial of yummy food and spent the week in a cosy 25°C incubator (see Figure 3).
Figure 3: Tray of crosses in yummy vials of food about to start their romantic weekend in the 25°C incubator.
Before the males met the females we had to selectively pick them from their vials. We had to first select males using similar methods that were required to choose the female virgins last week. However, because the males can’t get pregnant the procedure was a little simpler as the 8 hour waits were not required. Once the males were separated from the females of the same genotype, we selected the males that looked young (have more transparent bodies) and had intact wings, and put them in a fresh vial of food. We then selected the young and healthy females from the previously sorted vials and placed them into the vial with the men. These clearly labelled vials containing females and males of known genotypes were then placed in to the 25°C incubator for a week, allowing them plenty of time to mate and the females to lay eggs.
This process was repeated so that each of the 10 types of miRNA females were crossed with the 5535 males. We will look into how the offspring is sorted soon!