The overall goal of this procedure is to quantitatively measure the gene regulatory activities of CIS regulatory elements or CRE active during drosophila melan gastro metamorphic development. Begin with site-specific integration methods to generate lines of CREs Transgenically Incorporated as reporter transgenes. Using a stereo microscope, obtain transgenic specimens of the correct developmental stage.
Remove specimens from the outer pub and mount them on a glass microscope. Slide proceed to record the level and pattern of fluorescent reporter protein expression in whole mount samples by confocal microscopic analysis. Finally, quantify the level of fluorescent reporter protein expression for recorded confocal images using the Image J software tool.
These results assign a level of gene regulatory activity to assist regulatory element and facilitate further comparative gene expression studies on modified CRE versions. This procedure can help answer key questions and feels interested in gene regulation and how that gene regulation is encoding. DNA feels like developmental and evolutionary biology.
This procedure was developed to provide insights into cis regulatory elements that function during drosophila melanogaster metamorphic development. In principle, however, it can be applied to cis regulatory elements that function during other developmental stages and other fruit fly species, or even other model organisms. In order to expand transgenic lines, set up two vials with several male and female flies on alternating days.
Transfer the flies from one of the vials into a new vial. Repeat this for a week. By the end of two weeks, verify that the transgenic fruit flies range from larval to adult developmental stages.
Start staging specimens at the end of the third instar larval stage when the ER is formed. Note the specimen is non-mobile. The ER is softened white and the sphericals have averted specify this time point.
As zero HAPF. Distinguished males from females by the presence of bilaterally situated gonads near the midpoint of the body that appear as translucent circles for staging based upon length of metamorphosis. At zero HAPF, transfer specimens to a fresh vial using a moistened paintbrush.
Add a piece of Kim wipe and moisten with water to keep the specimens from drying out. Now, incubate the vial at 25 degrees Celsius until the desired HAPF. Alternatively, stage specimens by sorting based upon identification of the presence and position of several morphological markers as detailed in the accompanying text.
With lab tape, adhere a piece of packing tape to a dissection board with the sticky tape surface facing upwards. Wet a paint brush and apply moisture to ated specimens using a paint brush. Transfer specimens to a Kim wipe.
Transfer specimens to the packing tape and adhere with the dorsal surface facing up. Allow specimens to dry for 15 minutes. Progressively open up the ER using forceps.
Begin at the anterior or perm and proceed towards the posterior end. Then transfer pui to a drop of viscous oil on a microscope slide and evaluate the specimen microscopically for whole mount specimens. Use either the Forex or 10 x objective to bring the specimen into focus.
Using the confocal microscope software, adjust the excitation wavelength for the desired fluorescent protein to prevent photobleaching. Begin with a laser intensity of five to 10%If the signal is underwhelming, then increase the intensity as needed in order to improve the signal to noise ratio. For confocal images, enable software settings that average pixel measurements from replicate scans such as kalman averaging for quantitative comparisons of CRE activity uses C-section where fluorescence appears most intense.
Switch to a saturation warning lookup table and adjust the channel voltage gain and offset to settings where few pixels are saturated. If necessary, adjust the confocal aperture. Once the optimal settings are determined, run the ZS scan to obtain a series of images along the Z axis.
When the scan is complete, convert the image stack into a projection and save this image file in the TIF format. For quantitative comparison of CRE activities, use the same confocal settings for all replicate specimens and reporter transgene lines. For the experiment with the Image J program started open a TIFF image to be evaluated.
Click on the freehand selection button and outline the area of the specimen where quantitation is desired. To get a pixel value statistic, click on the analyze tab and then select measure. Record the mean pixel value score from the results box.
Also collect a second mean pixel value from a background region where the CRE is not active. To derive an adjusted mean pixel value, continue to generate pixel value statistics for replicate specimens. Then from the replica adjusted mean pixel values, determine the average regulatory activity for A CRE and also calculate the standard error of the mean.
This experiment uses intensity of the white rescue eye phenotype to make transgenic lines homozygous. A white gene mutant genetic background with an A TTP landing site sequence is utilized for site-specific transgene integration. Transgenic individuals, hemizygous and homozygous for the integrated vector are distinguished by the intensity of the rescued eye color phenotype by the number of copies of the integrated mini white gene.
Homozygous lines are then established by crossing male and female flies with the darkest eye color phenotype. Morphological markers are used to determine the metamorphic stage for drosophila during metamorphosis from larva to an adult fruit fly. The specimen transitions through a series of stereotypic morphologies that can be used to determine the sex and approximate the developmental stage.Here.
A wild type version of A CRE referred to as the dimorphic element drives high levels of EGFP reporter expression in the ASIC abdominal segment of female puy. A 13 base pair sequence within this CRE was found to be a binding site for the DSX transcription factor and the in vivo importance of this sequence is demonstrated by quantifying the regulatory activity for the CRE when this binding site is mutated. Quantitative analyses indicate that the mutation of this DSX site reduced the regulatory activity to 28 plus or minus 3%of the wild type expression in the context of the transgene insertion site used here.
Similar comparisons evaluate regulatory activities for intra specific CRE alleles or between autologous CREs from separate species, for example, compared to levels of EGFP in female segment. A six driven by the D Melin gastric Canin S strain autologous CREs for the species D stimulants and D willi possess regulatory activities equal to 75 plus or minus 4%and zero plus or minus 0%respectively. Followings procedure methods like RNA interference can be used with transcription factor genes to answer additional questions of how these transcription factor genes interact with CYS regulatory elements.
This would be indicated by the increase and decrease of fluorescent reporter protein expression that are driven by CIS regulatory elements After its development. This procedure paved the way for researchers in the field of gene regulation to explore the functional importance that cis regulatory element motifs and evolved mutations have upon gene expression.
