Modern genetic analysis relies heavily on techniques that facilitate the targeted removal of specific genes from cells, organs, or entire organisms. These knockout experiments help to reveal the function of the gene in its cellular context. The molecular morphological and physiological phenotypes of the knockout all provide clues about the role of the gene products encoded by the deleted sequence.
In model organisms such as mice, a common method of achieving a conditional gene deletion is by flanking the gene or exon of interest with locks P sites, which serve as recognition sequences for the Cree Recombinase enzyme. Irreversible excision of the so-called flocked allele is then catalyzed by Cree, thereby eliminating the sequence from the genome. Several different methods exist to introduce Cree Recombinase into the system, including those performed in vivo and those that deal directly with established cells.
For this particular application, we'll use an adenovirus to deliver Cree to fibroblast cells that have been isolated and cultured from a transgenic mouse carrying a flock allele. This method results in almost 100%efficiency of transgene delivery. When the virus is administered to the cells, it is internalized within an endocytic vesicle and transported to the nuclear envelope where its DNA, including the transgene of interest, can penetrate the nucleus through the poor complex.
The cellular machinery can then transcribe the injected gene and allow its protein products to act on the cell. In this case, the virally encoded Cree Recombinase enzyme excises the flocked allele from the host cell genome. We're from the Jones Lab at the University of Guelph in Guelph, Ontario, Canada.
In our lab, we're interested in the concept of signal transduction and what happens when you remove specific components of various signal transduction cascades. Today we're gonna be looking at a cascade related to actin organization. Signal transduction is accomplished by a series of biochemical components that transmit information and elicit phenotypic changes such as actin reorganization.
Our protein of interest is RAC one, a small gtpa that is important in mediating actin dynamics including cell adhesion, migration, and morphology. In this investigation, we will use the CRE LAX P system to remove this gene and confirm its role in the cell. Previously, fibroblast cells from embryonic mice with a flocked rack, one allele were isolated 12 days post fertilization.
These cells were then placed in liquid nitrogen for long-term storage, and we will begin by thawing a single vial of these cells representing one embryo. When performing cell culture experiments, it is important that a sterile environment is maintained. This involves wiping down the exposed surfaces of an approved flow hood, as well as spraying all reagent bottles and laboratory equipment that enter the hood with 70%ethanol.
Once the flow hood has been prepared, thaw the cells by immersing the vial in a beaker of 37 degrees Celsius water until the contents are liquid. Next, sterilize the vial with 70%ethanol and then add the cell suspension. Dropwise onto a 10 centimeter plate containing nine mils of DMEM, high glucose medium that has been supplemented with 10%fetal bovine serum and 1%penicillin.
Streptomycin gently rock the plate to disperse cells and place into the 37 degree incubator with 5%CO2, remembering to label the plate with the appropriate information. The cells typically reach 100%co fluency within 24 hours. At this point, we can passage the cells onto a new plate.
To do this, begin by aspirating off the old media. Add five mils of sterile PBS to rinse the cells and then remove this as well. Now add one mil of trypsin containing 10 millimolar EDTA.
Place the plate in the 37 degree incubator for approximately five minutes to allow cells to detach from the substratum. Return to the flow hood and add nine mils of media back to the tryps inized plate, thus diluting the cells one in 10, pipette the liquid up and down several times to break up cell clumps and then remove one mil of the suspension and add it dropwise to nine mils of fresh media in a new 10 centimeter plate. Again, gently rock the plate to disperse cells and place into the 37 degree incubator.
When cells have reached confluence, the next step is to seed them onto glass cover slips so that they can be viewed via microscopy. Prepare the cells using the same technique used for packaging up to and including adding trips into the cells and placing them in the 37 degrees Celsius incubator. While cells are lifting off the plate, prepare a six well dish with glass cover slips using one autoclaved cover slip per well.
Once cells have detached from the plate, add five to six mils of media back to the tryps inized cells. For the next stage of the experiment, cell counts will be conducted so that an appropriate number of cells can be seated for efficient virus infection. To count the cells combine 15 microliters of sample with an equal part of trian blue pipette up and down to mix and add 15 microliters of this solution to the hemo cytometer.
Dead cells will appear blue Under this preparation count only the live or colorless cells on the four by four grid to calculate the number of cells per microliter. Begin by counting cells in four separate grids, A through D, and taking the average. Then multiply this value by two to account for dilution in trian blue.
Since the grid holds one 10th of a microliter, multiply by 10 to express the figure as cells per microliter. Next, determine the required volume by dividing the desired number of cells by the cell concentration calculated previously. Add the calculated volume of the cell suspension dropwise to each well of the six weld dish.
Leave these cells in the 37 degree incubator overnight. The following day virus will be added to the cells. The den creek virus that obtained from Vector B labs was aliquot and stored negative 80 degrees Celsius.
Retrieve this virus and returned to the flow hood. Remove the six well dish from the incubator and bring it to the hood. Aspirate off the media and wash the cells with one mil of serum free DMEM.
Now add one mil of serum free media to each well for the addition of the virus. For the viral application, we use a multiplicity of infection or MOI of 500. Protocol optimization was previously conducted using various MOIs and we found that an MOI of 500 provided highly efficient infection rates with little or no effect on cell viability.
To calculate the MOI use the following equation. Begin by calculating the number of viruses required, which can be accomplished by multiplying the target MOI, in this case, 500 by the number of cells per well, or 30, 000 to determine the volume of virus stock that should be used, multiply the virus number by 1000 and divide this by the initial concentration of the virus. In this case, we will add 1.5 microliters of virus stock to each well that is to be infected.
Gently rock the plate to disperse the virus and place the plate in the 37 degree incubator overnight. The next morning, remove the virus media from the cells wash with PBS and add two mils of regular media to the cells. In this experiment, we found that phenotypic changes occurred in the cells in approximately 72 hours.
Cells stained using the Acton din were imaged using the Leica T-C-S-S-P five multi photon confocal laser scanning microscope at the Advanced Analysis Center at the University of Guelph. This revealed changes in morphology between infected and non-infected cells. Those lacking RAC one appear elongated and thin compared to the wide flatten cells that were not infected.
Here we've shown that the ADOC crease system is a robust method for efficient gene delivery to cells beyond the study of signal transduction. However, you can also use this method to knock out genes in experimental animals or maybe to label specific cell populations in an organism. The adenovirus system can also be used to introduce genes other than Cree to principle.
Advantages of using this delivery method include its high affection, efficiency and lack of integration with host cell DNA. However, the generation of a common adenoviruses can be labor intensive. The protocol described here therefore harnesses the power of the adenoviral system using the previous developed ocre virus with our flocked RAC one mes through this conditional knockout, we have shown that.
So morphology is indeed partly dependent on the small GTP as RAC one. So in conclusion, we from the Jones Lab would just like to say thanks for watching our video and good luck in all of your future research.
