The overall goal of this procedure is to determine if two genes are functionally redundant for specification of a defined cell lineage. This is accomplished by first defining the loss of function phenotype of each individual gene using gene specific morphos injected into developing zebrafish embryos. The second step of the procedure is to develop an assay for quantifying cell lineage specification or differentiation.
For example, using a reporter fish strain, as we will show here. The third step of the procedure is to combine two morph FENOs to target the loss of function for both gene simultaneously. The final step of the procedure is to evaluate the phenotype caused by double knockdown.
Ultimately, results can be obtained that show loss or gain of function for cells of a specific lineage through examination of the lineage reporter or analysis of expression for lineage specific marker. The main advantage of this technique over existing methods like combining knockouts in the mouse model, is that two or even three genes can be targeted in the fish model simply by combining validated morphos in a single experiment. Over 100 embryos can be injected and phenotypes evaluated over the course of several days.
This method can help answer key questions. In developmental biology such as defining the transcriptional pathways that control cell fate, key regulatory pathways are often represented by small families of related genes that are functionally redundant. Their role is therefore not evident from mouse knockout studies due to compensation from Sister Genes.
Although this model can provide insight into zebrafish embryogenesis, the results can be applied to other systems such as mouse, since the same genetic programs are typically well conserved throughout evolution. Prepare microinjection plates before injecting embryos. Pour approximately 12 milliliters of 2%agros in system water, which is clean water taken directly out of the fish tank system into the inverted lid of a 100 millimeter Petri dish.
Rest two microscope slides at approximately 45 degree angles into the two sides of the lid. When the agros has solidified, gently pull the slides away from the lid, creating troughs where the embryos will rest during injection morph fo, no stalks are kept at room temperature at a concentration of one millimolar and sterile distilled deionized water Before injecting. Incubate morphos stocks at 65 degrees Celsius for five minutes to ensure that they're completely in solution.
Allow the stocks to cool to room temperature. Each newly designed morpho must be empirically validated for activity and embryo tolerance. In micro centrifuge tubes, start with one to two serial dilution of the stock morpho in distilled deionized water yielding working concentrations of one millimolar 0.5 millimolar 0.25 millimolar and 0.125 millimolar.
Under a dissecting scope, use suction to front load a calibrated injection needle that has been attached to the micro manipulator and properly positioned. Load the needle with approximately one microliter of the lowest concentration morpho solution. Use a transfer pipette to move fertilized one cell embryos into the troughs of the microinjection plate.
Use the pipette to remove excess water from the plate so that the embryos fall to the bottom of the trough. Position the injection plate under the microscope, descend the needle through the corion and into the yolk. Inject each embryo before removing the needle and repositioning the injection plate to access the next embryo.
When learning to inject, it may be helpful to use a vital dye to visualize the injection into the cell as shown here, transfer the embryos back to a 100 millimeter Petri dish with system water culture them at 28.5 degrees Celsius. Expel any remaining morpho solution from the needle, and fill it with the next highest concentration morpho for injecting the next batch of embryos at an appropriate stage of development. Examine the embryos for phenotypes at each injected morpho concentration.
The threshold dose for each morpho is the lowest dose at which there is a defined reliable phenotype. Multiple rounds of titration may be necessary to define inaccurate threshold. To look for a genetic interaction between two distinct genes.
Prepare a mixture of two morphos of interest each at its respective threshold concentration. It is important to keep in mind the embryos may not tolerate more than 20 nanograms of total morpho per injection. Inject the embryos in the same manner as before.
Keep injection volumes constant between experimental and control groups, which should include sets injected with each morpho alone under the dissecting microscope. Monitor injected embryos immediately after injection and several times throughout the day, use a transfer pipette to remove any dead or dying embryos. Since these can compromise the viability of the remaining morphines with a transfer pipette moves, sedated or euthanized embryos at any time.
Point to a depression slide for observation. For photography. Use sharp forceps to remove the corion if necessary.
Stabilize the embryo in a drop of 3%Methyl cellulose screen embryos for a distinct phenotype, unique to the combination of morphos as opposed to greater penetrance of the phenotype of single morphines. Here are several wild type embryos when injected with morphos at threshold. For gata five, the typical phenotype is cardio bifida or two hearts, because the progenitors failed to fuse at the midline.
Notice the GFP positive cardiomyocytes indicated by the arrows. The phenotype for six morphines includes misshaped hearts that fail to loop properly. These embryos also develop GFP positive cardiomyocytes.
However, the embryos injected with a combination of both gata five and gata six morphos shown here. Exhibit total loss of cardiomyocyte development indicating that either gata five or gata six must be expressed for cardiomyocytes to develop. The two genes are functionally redundant for cardiomyocyte specification.
While attempting this procedure, it's important to first fully validate your morphos and be as certain as possible that they represent a true knockdown of single gene target Following this procedure. Other methods like combining conditional mouse null alleles can be performed in order to show that the same genes function. Likewise in mammals.
After watching the video, you should have a good understanding of how to determine if two genes are functionally redundant during embryogenesis.
