How to know chromosomal location of a transgenic construct by using a genetic cross ?

By | July 1, 2021
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When we generate a new transgene, the first thing which needs to be done is to map its chromosomal location. This can be done at molecular level by performing Inverse Polymerase chain Reaction (iPCR) and subsequent sequencing.

It is also possible to efficiently map the P-element insertion (transgene) by doing a simple cross genetic crosses and observe for two generations. This method allows the fly researcher to know the chromosomal location of the transgene and also balance the new insertion in one go.

We will see how that is done: ( Note – For these crosses we are ignoring 4th chromosome as it is very tiny and possibility of the transgene landing on that chromosome is very minimal. However we can also trace presence of transgene on 4th chromosome by this genetic method)

First we need to select a single male transgene and cross it to few female virgins from white eyed double balancer stock ( Pin / Cyo ; TM2 /TM6Tb ).

If in the next generation only females’ show eye color and all males are white eyed, one can definitely say that the insertion is on X chromosome.

Since we have used a red eyed male (eye color in these flies will come only from the transgene and that is due to the mini white gene cloned into the p element transformation vector used for generation of new transgene ) for the cross and all thet progeny in F1 generation showed red eyed females, which is only possible if the insertion is on X chromosome.

The male parent will contribute its X chromosome to female embryos and Y chromosome to males in next generation (F1). So the red eye color from transgene, which is inserted on X chromosome of male parent can be only be transferred to female embryos in F1 generation and hence all females files are red eyed.

Only females in F1 generation show eye color – Insertion on X chromosome

However if both males and females exhibit eye color, then the transgene can be on either 2nd or 3rd or 4th chromosome. To confirm on which chromosome the transgene is actually inserted :

We need to take few males with eye color with Tb and pin markers ( Pin marker can be used even though its not a balancer as recombination is not possible in Drosophila males) and cross them to Curly, TM2 females with eye color and observe the flies in F2 generation.

Imagine the insertion is on second chromosome then we except all flies with Pin and Cyo markers will be white eyed.

Drosophila being a diploid organism and both sister homologs of second chromosome are occupied by Pin and Cyo chromosomes ( as in Pin / Cyo flies).

Then the transgene present of second chromosome in F1 flies cannot be accommodated as both the chromosome slots in diploid organism are occupied by Pin and Cyo chromosomes.

Hence all Pin / Cyo flies will be white eyed, and this proves insertion of the transgene on second chromosome.

Insertion of transgene on second chromosome

In case of both males and females flies bearing Pin and Cyo markers in F2 generation show eye color then the insertion of transgene will be on third chromosome. This can also be verified further when all flies in F2 exhibiting TM2 and Tubby markers (black body -ebony flies) are white eyed. The transgene present of third chromosome cannot be accommodated in flies bearing both TM2 and TM6Tb chromosomes, hence the reason for white eyes in flies with both the third chromosome markers.

Insertion of transgene on third chromosome

If both males and females, Pin / Cyo ; TM2 /TM6Tb fly shows eye color then the insertion is on 4th chromosome.

Balanced stocks can also be generated simultaneously by collecting Pin/ Cyo ; Transgene (eye color) / TM6Tb flies in above cross and Eye color / Cyo ; TM2 / TM6Tb for the second chromosome insertion cross.

Identification of markers for third chromosome balancers :

For identification of different fly markers please visit

John Roote and Andreas ProkopG3: GENES, GENOMES, GENETICS February 1, 2013 vol. 3 no. 2 353-358;