User:Lmohamm9/sandbox

= Ectopic expression = Ectopic expression ''is the expression of a gene in a tissue where it is not normally expressed. Ectopic expression also refers to the expression of a gene at a point in the cell cycle or in an organism's development when it is not normally expressed. This can be caused by a disease, or it can be artificially produced as a way to help determine the function of that gene.'' Gene up-regulation may mediate the development of structures in abnormal locations, resulting in ectopic expression.

Ectopic expression systems
Ectopic expression of a gene can be done by introducing a transgene with a modified promoter into the target organism (transient or stable transfection) or by using the Gal4/UAS system. As a result, use of ectopic expression systems can introduce a new phenotype which may be lethal. If an extreme phenotype is to be expected, a promoter which is inducible should be used.

Traditionally, ectopic expression of a gene using Drosophila melanogaster involves mutant generation via dominant mutations. This allows for the over expression and/or misexpression of the final functional gene product. Elaborate studies by the D. melanogaster research community gave rise to multiple ectopic expression systems available for use.

With all D. melanogaster ectopic expression mechanisms, a promoter designed for the gene of interest is utilized to initiate transcription. The promoter results in either constitutive or regulated gene expression. In order to observe the effects between wild type and ectopically expressed genes, a transgenic line must be produced.

D. melanogaster polyubiquitin and EF-1α promoters
Polyubiquitin and EF-1α promoters from D. melanogaster, have been utilized for uniform ectopic expression. EF-1α promoters allow for elevated levels of transcription while maintaining uniform expression. On the other hand, use of polyubiquitin promoters such as the polyubiquitin-lacZ can be limited. The polyubiquitin-lacz promoter allows for uniform expression in early embryos and imaginal discs, not other developmental stages.

Pwum2, a P-element polyubiquitin vector, has been proven effective in D. melanogaster ectopic expression. Pwum2 includes a polyubiquitin promoter combined with a myc tag. The vector itself includes two restriction enzyme sites (KpnI and Not1). The tag has a significant role in ectopic expression systems, allowing for the detection of encoded proteins in transformed organisms.

Heat shock ectopic expression
Heat shock promoters, promoters from heat shock proteins (HSP), have been used in D. melanogaster to regulate gene expression. Compared to higher organisms, D. melanogaster temporal temperatures can be regulated, this allows manipulation of ectopic expression levels. In addition, altered durations of heat treatments can be used to modify levels of ectopic expression.

There are three disadvantages when it comes to the heat shock ectopic expression system. Firstly, the transcription levels for some heat shock promoters is low under non-heat shock conditions. This is detrimental to the cell if the gene encodes a toxic product. Secondly, it is difficult to study tissue-specific functions when heat shock promoters are expressed in all cell types during an experiment. Lastly, under heat shock conditions the heat shock promoter is active, however, endogenous cellular gene expression is disrupted. Decreased expression of specific regulatory genes due to heat treatment may result in mutant D. melanogaster phenotypes.

Agouti gene
The agouti gene in mice is responsible for the pigmentation of mouse coat patterns and hair follicle development. Ectopic expression of this gene is variable in mice and has been associated with the predisposition of tumours and obesity. The mechanism of ectopic expression involves the epigenetic regulation of IAP (Intracisternal A-Particle), an insertion element located upstream of the agouti gene. When the IAP is demethylated in mice, ectopic expression is driven from a cryptic promoter encoded by the IAP insertion. Minimal ectopic expression of the agouti gene results in mice with a brown fur coat, maximum ectopic expression of the agouti gene results in mice with a yellow fur coat.