In conclusion, I have discussed the basic components of the molecular and genetic toolkit that have been shown to work for ectopic gene expression in Drosophila testes. We find expression of targeted mRNAs and eGFP-tagged fusion proteins is typically first detected in 8-cell spermatogonial cysts, peaks in early-mid spermatocytes and declines as spermatocytes mature Fig. Undoubtedly there are many more drivers that could be used to express ectopic genes in the somatic cells of the testis, however the male genital tract expression of most Gal4 lines that are available over stocks listed in Bloomington Drosophila stock center has not been characterized. It has been used in numerous studies, including the demonstration that human BOULE is capable of rescuing the meiotic defects caused by boule loss of function in Drosophila testes. Organizing activity of wingless protein in Drosophila.

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Some genes are activated as cells become spermatogonia, and presumably some are inactivated at this transition, but there is plasticity in the system as spermatogonia can, under appropriate conditions, de-differentiate and revert to stem cell identity.

GAL4/UAS system – Wikipedia

Paradigms and lessons from Saccharomyces cerevsiae “. Disadvantages include a lack of flexibility, since a new construct is needed for every expression pattern tested. There is limited post-meiotic transcription in Drosophila melanogaster spermatids, and a small set of genes are transcribed at a higher level in these cells than ga4l earlier stages.

Geneticists have created genetic varieties of model organisms typically fruit fliescalled GAL4 lineseach of which expresses Yal4 in some subset of the animal’s tissues. This cell becomes the head cyst cell, remains relatively small and covers the spermatid heads, while the tail cyst cell elongates to ensheath the full length of the spermatid bundles.

Until recently it was thought that virtually all transcription in the Drosophila germline was pre-meiotic, however it is now clear that spermatids are transcriptionally active. Here I will summarize ectopic expression systems that are known to work for the testis, and provide advice for selection of the most appropriate expression system in specific experimental situations.


The dynamics depends on the stability of the ectopic protein, and we have examples where the ectopic protein is detected in late-elongation stage spermatids Fig. These constructs could be used to gap4 ectopic proteins at the ends of elongating spermatids by simply replacing the schuy or c-cup ORFs with the gene of interest. Also on the wish list would be the testing of alternative bipartite expression systems in the testes. Dramatic cell growth occurs during the spermatocyte stage, before entry into the meiotic divisions.

This article has been cited by other articles in PMC. Hsp83 -based promoters are also useful for ubiquitous expression.

Experimental genetics using Drosophila melanogaster has advanced dramatically with the advent of systems for ectopic expression of genetic elements in specific cells. Conclusions and Future Perspectives Why some constructs work while others don’t Ectopic expression constructs in Drosophila originate via two independent routes. It has also ggal4 adapted to study receptor chemical-binding functions in vitro in cell culture. The disadvantage is that at least three independent insertion sites are required, nanks the lines must use different and independent ga4l sites, and creating the desired final organisms needs more than a single cross.

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For this the betaTub85D constructs have proved very useful, for example, knock down of fan expression, although not complete, was achieved with a fan hairpin cloned into a betaTub85D expression vector. After elongation the spermatids undergo an individualisation process and finally mature sperm coil at the base of the testis before passing into the seminal vesicle for storage until mating.

Brand AH, Perrimon N. For high level expression of ectopic proteins in spermatids it would probably be better to use a vector based on a gene expressed in primary spermatocytes, but not translated until spermiogenesis as discussed above. The caveat here is that the ORFs might contain regulatory signals, which would be missing in the transgenes. Intriguingly, HSF is also detected in spermatid nuclei, raising the untested to my knowledge possibility that the heat shock system could be used to activate a burst of post-meiotic gene expression.


Instead I tested drivers based on genes expressed independently of the meiotic arrest loci, and earlier in primary spermatocytes.

B Different Gal4 driver lines can be used to drive downstream gene expression with spatial and temporal control. Some might work, but we don’t know about them. Thus exactly what has, and has not, been tried is not available for analysis.

Translational control in spermatogenesis. Expressional and functional analysis of the male-specific cluster mst36F during Drosophila spermatogenesis. In the cells that are producing GAL4, however, the UAS is activated, the gene next to it is turned on, and it starts producing its resulting protein. Basic expression constructs suitable for testes germline or soma.

In the offspring the selected cells, and only those cells, will contain channelrhodopsin and can be triggered by a bright light.

FlyBase Recombinant Construct Report: P{nos-GAL4.U}

Extra considerations for RNAi induction All the systems described above are generally applicable, and not restricted to expression of a single type of ectopic element. Constructs for ubiquitous expression There are a range of experimental situations where expression in the male germline is essential, and expression in the soma is either also desired or at least is not detrimental.

Because there is only limited transcription in spermatids the transcripts encoding proteins required for post-meiotic processes are almost all abundantly transcribed in spermatocytes.