Hi, I'm Erica Kgi from the laboratory of Shannon Fisher in the Department of Cell and Development Biology at University of Pennsylvania. I'm Chris Weber, also from the Fisher Lab. And I'm Shannon Fisher.
Today we'll show you a procedure for analyzing the function of enhancer elements using zebrafish transgenesis. We use this procedure to study transcriptional regulations of genes important in skeletal development. So let's get started.
To identify non-coding sequences surrounding candidate genes, we use the algorithm fast cons, which is accessible as a track on the uc SC genome browser. We are primarily interested in genes expressed during skeletal development, but the same approach can be used to study any genes expressed in early development. In the example shown here, we have identified conserved sequences.
In the interval surrounding the human bumper gene, we examine the entire interval to the adjacent genes and determine a low size specific log score cutoff corresponding to approximately the top 5%of non-coding sequence. After selecting sequences, design primers using primer three to amplify each sequence from human genomic, DNA primers should be selected to amplify the conserved region plus 30 or more base pairs on either side. Amplify the region of interest with high quality polymerase using human genomic DNA, use standard laboratory molecular biology procedures to clone the amplified product into an expression vector.
The vector used in this protocol is PG wc FOS GFPA gateway. Destination vector. Following vector creation told to transpose.
Enzyme required for transposition is first transcribed in vitro using the M message M machine kit from Ambion and the PGB 600 plasmid prior to experimental injections. Test each batch of transposes for efficacy by injecting with a known enhancer such as the mouse socks. 10 enhancer.
To prepare for injections, pull the needles used for microinjection from 1.2 millimeter od filament capillary glass on a Sutter P 97 micropipet puller. Use a program that yields a strong tip with a sharp taper to penetrate intact corion's with a clean razor blade and micrometer slide. Break the tips by hand under a stereo microscope to an outer diameter of approximately 15 micrometers.
The needles can be stored for a day in a covered holding dish. Maintain zebra fish population on a regular light dark cycle with 14 hours of light under standard conditions the day prior to performing micro injections. Prepare fish for timed matings in small breeding tanks.
Place three females or two males per tank in parallel rows on the morning of the micro injections shortly after the light cycle begins. Combine males and females in clean system treated water for egg production. Check on the fish frequently and collect the eggs within a few minutes of production to get well synchronized batches.
Maintain egg production for two to three hours by continuing to combine fresh groups of fish throughout the morning with a wide bore five and a quarter inch glass past pipette fitted with a latex bulb. Sort the collected embryos into 60 by 15 millimeter Petri dishes, partially filled with embryo medium in groups of 50 embryos. Mark the time collected on the lid of each dish.
For each clutch of embryos collected, save a dish of 50 embryos for the unin injected control between egg collections. Prepare fresh injection solutions by mixing the following ingredients in a micro fuge tube and store on ice. Lay the injection needles in holding dishes and fill with 1.5 to two microliters of injection solution.
By pipetting 500 nanoliter drops onto the wide end of each needle. The liquid will be drawn to the tip through capillary action. Prepare at least two needles for each injection solution.
Load the filled needle into the handheld needle holder of a pneumatic pico pump or similar pressure injector. Configured and connected to a nitrogen tank per manufacturer's instructions. Calibrate injection volumes by measuring the diameter of droplets expelled into mineral oil on a micrometer slide, typically an injection time of 120 milliseconds.
With a pressure of 20 PS, I will yield a droplet of approximately one nanoliter using a stereo microscope at six to 10 x magnification. Hold the embryo in place with a pair of fine forceps and push the injection needle with steady pressure through the corion into the yolk of an embryo at the one cell or early two cell stage, position the needle tip in the yolk just below the blastomeres expel approximately one liter of injection solution and withdraw the needle for each construct we inject greater than or equal to 200 embryos after injection are completed. Sort the embryos by removing unfertilized eggs, damaged embryos, and failed injections or embryos with no phenol red in the blasphemers.
We then incubate the embryos and embryo medium at 28.5 degrees Celsius until the appropriate time for observations. Now we'll show you some representative results of enhancer elements that produce tissue specific patterns of GFP expression in the zebrafish embryo. We study genes expressed in bone or cartilage during.
Here's an example of an enhancer element upstream of the acan gene that regulates expression in cranial facial cartilage at three days post fertilization. Here is another enhancer this time from the bumper gene that regulates expression in bone cells. At five days of the many enhancers we have identified, we find little correlation between location relative to the gene and regulation of expression.
When choosing sequences to test, it is important to remember that an enhancer can be hundreds of kilobases upstream or downstream of a gene or in one of the introns as seen in this embryo. A substantial portion of sequences do not appear to regulate tissue specific expression. These often show very little fluorescence or may have a few scattered cells in two common sites for ectopic expression, the epidermis and skeletal muscle.
We've just shown you how to identify potential enhancer elements through sequence conservation and test their function through Mosaic transgenesis In zebrafish. When doing this procedure, it's important to examine the quality of your DNA and RNA for injections. Also be, be sure to examine all of your injected embryos carefully, especially if you only expect expression in a small group of sales.
So that's it. Thank you for watching and good luck with your experiments.
