How to balance and generate new fly stocks for genetic crosses

By | June 19, 2021
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Drosophila melanogaster was introduced as a model organism more than a century ago. However it was the pioneering work of Thomas Hunt Morgan and his graduate students Sturtevant and Bridges have revolutionized the field of Drosophila genetics and laid the foundation for the present day Invertebrate powerhouse for studying development, physiology and behavior. Since more than 70% of known human disease genes have a counterpart in Drosophila genome, It makes fruit flies an excellent in vivo system to decipher proteins and its interacting partners responsible for many human diseases. The rapid growth in availability of genetic , molecular and biochemical tools allows us to dissect cellular and molecular mechanisms underlying various pathways related to development and disease ,this will eventually lead to discovery of novel drugs for healing the diseases.

Importance of balancing and generating Fly stocks with different variants on multiple chromosomes

The construction of a huge building depends on a strong foundation. Similarly performing a complicated genetic crosse to decipher biological puzzles under study, requires good planning and 100% accuracy in generating fly stocks. If one makes a mistake while building of stocks then the whole interpretation of the end result will be compromised.

In a Fly lab, Collection of virgins females and performing genetic crosses is a part of daily routine for a fly researcher. These genetic crosses involve various gene mutations, transgenic constructs and chromosomal deficiencies ( fly variants) which are either studied in isolation or in combination of others to extract maximum information related to genes under study. The different fly variants forms an important part of research tools as they provide vital means to understand the function of genes.

To understand the role played by these fly variants in deciphering the gene function , we can take an example. Suppose we have a isolated a novel mutation affecting a gene ( By CRISPR or P- element mutagenesis) , which in homozygous condition shows disruption of eye primordial cells in the developing embryo and also affects the expression pattern of known functional proteins in eye. This indicates possible role of our newly discovered gene in eye development. In order to prove our hypothesis we should do a rescue experiment to see reversal of our loss of function phenotype, by expressing ectopically the wild type version of the same protein in eye cells (with complete genome sequence of Drosophila available it is lot easier to know sequence of any gene for generating transgenic constructs) .

To perform this actual experiment in a fly lab we need to get mutation and UAS- construct of the gene in one fly and and then cross to GAL4 driver stock. This requires careful designing of genetic cross and vigil should be kept at every step of stock building to obtain the desired result. Theoretically it all looks so simple but it requires lots of experience and skill along with planning for several generations ahead.

Balancing fly variant

  • The first step involved in case of generation of any new fly variant or new stock ordered from stock center is to make it stable using a appropriate balancer chromosome.
  • This is the pivotal step in stock building as it will prevent the fly variant from undergoing any unwanted recombination and also allows us to identify and track the chromosome on which fly variant is present during genetic crosses.
  • The presence of unique chromosomal arrangement on balancer chromosomes and easily identifiable dominant markers allows the researcher to perform the desired function of making new fly variant stable and traceable in a single go.
  • We will try to understand the above step of balancing a fly variant by taking a example from our previous post. In that post we wanted to perform a large scale genetic screen and for that purpose we generated a recombinant fly line of two fly variants act5c-GAL4 and Tubp-GAL80ts present on second chromosome.
  • This recombinant of act5c-GAL4 – Tubp-GAL80ts needs to balanced first so that we can get it in combination with the mutation in gene of interest present on third chromosome in one fly to build a stock required for genetic screen.
Fig : 1 Balancing of recombinant on II chromosome
  • To balance any fly variant, the first step involves is crossing to respective balancer chromosome. Just to remind , Drosophila is a diploid organism meaning it has two homologous sets of chromosomes and to briefly mention fly nomenclature : homologous chromosome is separated by a slash and a ” ; ” is used to separate two chromosomes.
  • In the above Fig. 1 since act5c-GAL4-Tubp-GAL80ts recombinant line is on second II chromosome we need to use balancers for II chromosome ( CyO).
  • The balancing of the recombinant chromosome will work by crossing to Pin / CyO fly stock but since we ultimately need to get mutant of gene of interest also on third chromosome, it is advisable to cross it to the double balancer fly line (Pin / CyO; TM2 / TM6B).
  • For step by step balancing procedure we just need to follow from F2 generation in Fig:1 , where we have a single recombinant chromosome balanced over CyO chromosome plus a wild type (+) chromosome / TM6B balancer for III chromosome.
  • This single fly is crossed to pin / CyO ; TM2 /TM6B double balancer fly line to collect enough number of males and females of genotype act5c-GAL4-Tubp-GAL80ts / Cyo ; TM2/ TM6B .
  • This balances the recombinant variant on II chromosome and also will have balancer on III chromosome to ensure that the incoming mutant gene of interest is also balanced in a single cross in next generation.

Step by step balancing:

  • While balancing it is important to remember that, we have CyO chromosome in both parents and we need to select CyO coming from the male parent and not from the female parent in F3 generation or else we will lose recombinant chromosome.
  • To do this we need to select for eye colour (coming from act5cGAL4- Tubp-GAL80ts chromosome due to mini white in transgenes) and by doing this we will be selecting against CyO chromosome from female parent . This is based on Mendle’s law of segregation which states that sister chromatids segregate from each other during meiosis and in our example only one sister homolog of second chromosome is passed on to offspring’s in next generation.
  • So now we have few eye color flies ( recombinant chromosome) and if we also select for CyO chromosome from male parent ( by opting for curly winged flies and against Pin – sharp, short thoracic bristles ) to ensure they are balanced on second chromsome again in F3 generation to prevent recombination.
  • This way we can select for a chromosome either by selecting for a marker of interest ( eye color) or by selecting for and against a particular marker present on balancer chromosome ( selecting for curly wings or by selecting against pin bristles).
  • Similarly we can select for TM6B ( short body and humeral bristles on shoulder of fly) chromosome from female parent in F2 and TM2 ( bristles on haltere) from male parent to completely balance the recombinant line with balancers on II and III chromosomes (act5c-GAL4- Tubp-GAL80ts / CyO ; TM2 / TM6B ) .

So all male and female flies selected in F3 should have eye color, curled wings and non pin bristles ( II chromosome) along with bristles on haltere , short body with humeral marker and black body color due to presence of ebony marker on both TM2 and TM6B balancers .

This ensures we have a 100 % authentic recombinant chromosome of act5c-GAL4- Tubp-GAL80ts balanced over CyO chromosome and with additional double balancers on third chromosome.

Generation of new stock for genetic cross :

  • In this section we will look into details about building a new fly line by combining chromosomes present in two different fly stocks.
  • In one fly we have act5c-GAl4-Tubp-GAL80ts on second chromosome and in another fly we have mutation in gene-x ( Mut-X) on III chromosome.
  • In step by step manner we will see how to build a new stock with both these fly variants in one fly line or stock.
  • Since we already have act5c-GAl4-Tubp-GAL80ts / CyO ; TM2 / TM6B balanced line , we just to need work with mutation in Gene-x on III chromosome to accommodate act5c-GAl4-Tubp-GAL80ts II chromosome.
  • To do that we should first generate a fly line with balancers on II chromosome along with balanced Mut-X on III.
  • First the Mut-X line should be crossed to double balancer line Pin / CyO ; TM2 / TM6B to collect + / Pin ; Mut-X / TM6B males and + / CyO ; Mut-X / TM6B females in F1 generation.
  • These flies from F1 should be crossed to select Pin/ CyO ; Mut-X / TM6B flies to generate the stock of Mut-X with balancer on II chromosome.
  • One thing to remember is to see that all flies should have both pin bristles and curly wings marker in F3 generation flies. We don’t have to worry about third chromosome as both Mut-X and TM6B in homozygous condition are embryonic lethal and all eclosing flies in F3 will be of Mut-X / TM6B genotype.

It is important to have a marker and balancer on II chromosome even though we have fly variant Mut-X only on II chromosome. By having a marker and balancer on II chrosoome allows us to accommodate and balance with ease the act5c-GAl4-Tubp-GAL80ts chromosome in next cross, this is why planning ahead for generations is quite important in fly genetics. This also ensures that the act5c-GAl4-Tubp-GAL80ts chromosome is always balanced to prevent further recombination in any generation during stock building process.

Final Cross in stock building:

In this final step we need to cross both the individual stocks generated previously : act5c-GAl4-Tubp-GAL80ts / CyO ; TM2 / TM6B and Pin/ CyO ; Mut-X / TM6B flies. In F2 we have to select non pin, Curly wings , non ebony, eye color flies to get act5c-GAl4-Tubp-GAL80ts / CyO ; Mut-X / TM6B or TM2 fly stock required for genetic screen.

Hints for building stocks quickly

Fly variants with double balancers : Always keep an extra copy of important fly variants related to your project with balancers on other chromosomes ( if your fly variant is on II chromosome, it is always better idea to have stock with balancers on X with fly variant and also another one with fly variant plus third chromosome balancers

Maintain double balancers stocks in big bottles : It is advisable to always maintain the following double balancer lines in decent amounts – 1) null mutation of any gene / FM7 ; Pin / CyO , 2) null mutation of any gene / FM7 ; TM2 / TM6B and 3) Pin/ CyO ; TM2 / TM6B