Name: Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts (Video) | JoVE Journal
Uploaded: 2010-09-09T17:00:00.000+00:00
Duration: 11 min
Description: Learn adenoviral Cre delivery for conditional gene knockout in primary mouse embryonic fibroblasts. This method achieves nearly 100% transgene efficiency to study Rac1 function in actin dynamics.

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5 minutes

The authors would like to thank Dr. Rizaldy Scott at Mount Sinai Hospital in Toronto, Ontario for his gift of the primary Rac1flox/flox mouse embryonic fibroblasts. This work was supported by operating grants to NJ from the Canadian Institutes of Health Research (CIHR, MOP 2009-93526 ) and the Natural Science and Engineering Research Council of Canada (NSERC, RG 327372-06). SH and MW are supported by CGSMA and CGS-M awards from CIHR and NSERC, respectively.
Steve Hawley and Melanie Wills contributed equally to this paper.

Thawing cells
<ol>
 <li> Obtain frozen mouse embryonic fibroblasts (MEFs) (generated previously from individual embryo primary cultures) from liquid nitrogen. </li>
 <li> Thaw cells by immersing vial in 37°C water with swirling until contents are completely thawed.</li>
 <li> Add 9mL of DMEM that has been supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin to a 10cm cell culture plate.</li>
 <li> Add the thawed cell suspension drop-wise to the plate, rock the plate gently to disperse cells and place plate in a 37°C, 5% CO2 incubator overnight. Note that if cells are sensitive to DMSO (from the freezing process), initial media can be replaced with fresh media after cells have adhered to the plate (approximately four hours post plating). Also note that confluence of the cells after thawing depends on the number of cells frozen, the recovery rate of the cells after freezing, and the growth rate of the cells.</li>
</ol>
Passaging cells
<ol start="5">
 <li> When cells have grown to essentially 100% confluency (ie: the next day), aspirate off the media and wash cells with 5mL of sterile PBS.</li>
 <li> Remove PBS and add 1mL of trypsin containing 10mM EDTA and place plate in incubator for approximately 5 minutes to allow cells to detach from plate.</li>
 <li> When cells have detached, add 9mL of media to the trypsinized cells, pipette 2-3 times to disperse clumps, add 1mL of this suspension drop-wise onto a new plate containing 9mL of media, and place this into the 37°C incubator overnight. The presence of FBS in the media will inactivate the trypsin – alternatively, cells can be washed first to dilute out the trypsin.</li>
</ol>
Counting and plating cells onto cover slips
<ol start="8">
 <li> Remove cells from incubator, wash and trypsinize as before, except this time add 5-6mL media back to the trypsinized cells instead of 9mL. </li>
 <li> Pipette cells thoroughly to ensure complete dissociation into a single cell suspension, and remove 15μL to combine with 15μL of trypan blue in a separate microfuge tube. </li>
 <li> Mix this cell/ trypan blue suspension by pipetting up and down several times, and add 15μL to the hemocytometer. </li>
 <li> Under the microscope, dead cells will appear blue. Count the number of clear/colourless (living) cells in one of the 4X4 grids on the hemocytometer. Repeat this count on the three other 4X4 grids (denoted A, B, C and D) and use the following equation to calculate the number of cells per μL of suspension: 
 <img src="/files/ftp_upload/2160/2160eq0.jpg" alt="Cell count formula equation, calculating cells per µL using average of A, B, C, D; multiplication factor." data-altupdatedat="1788235205000" data-alt="Equation 1"></li>
 <li> Through protocol optimization, we have determined that plating 30,000 cells results in appropriate cell density for visualization at the conclusion of the experiment.</li>
 <li> To calculate the volume of cell suspension needed for 30,000 cells, use the following equation: 
 <img src="/files/ftp_upload/2160/2160eq1.jpg" alt="Cell concentration formula, volume calculation for cell culture; equation: volume (µL)=30,000/cells per µL." data-altupdatedat="1788235205000" data-alt="Equation 2"></li>
 <li> Now place a sterile glass cover slip into each well of a six well dish and add 2mL of media to each well, and then add drop-wise from your cell suspension the volume required for 30,000 cells into each well. Note that glass cover slips are used for this experiment as we will be visualizing the cells using fluorescence microscopy at a later time point; not all applications will necessarily require microscopy, and thus cover slips may not be required.</li>
</ol>
Virus Transduction
<ol start="15">
 <li> After several hours or overnight (sufficient time to ensure that the cells adhere to the cover slips), remove media and wash cells with 1mL of serum-free DMEM. </li>
 <li> Add 1mL of serum-free media to each well for the addition of the virus. For this experiment, adeno-Cre virus was obtained commercially from Vector Biolabs.</li>
 <li> Previous protocol optimization has shown that a multiplicity of infection (MOI) of 500 provides highly efficient gene transduction into primary MEFs with little or no effect on cell viability. MOI can be calculated as follows: 
 <img src="/files/ftp_upload/2160/2160eq2.jpg" alt="Equation for calculating virus quantity, Desired MOI × number of cells = viruses needed, formula." data-altupdatedat="1788235205000" data-alt="Equation 3"> 
 
 <img src="/files/ftp_upload/2160/2160eq3.jpg" alt="Virus stock volume calculation formula; equation for lab experiments and virology research." data-altupdatedat="1788235205000" data-alt="Equation 4"> </li>
 <li> Add the calculated volume of virus directly to each well that is to be infected, gently rock the plate to disperse the virus and place cells into the 37°C incubator overnight. </li>
 <li> The next morning, remove the virus-containing media from the cells and wash cells in 1mL sterile PBS and then add 2mL of regular media to each well.</li>
 <li> In the case of the Rac1 cells exemplified in this experiment, morphological changes occurred in the cells at approximately 72 hours post transduction.</li>
</ol>
Representative Results
Cells were visualised by staining with the actin dye phalloidin (for imaging purposes only) and imaged using the Leica TCS-SP5 multiphoton confocal laser scanning microscope at the Advanced Analysis Centre at the University of Guelph. Figure 1 shows the contracted and elongated morphology of the Rac1flox/flox cells that were exposed to adeno-Cre virus compared to the same cells not exposed to virus.
<img src="/files/ftp_upload/2160/2160fig1.jpg" alt="Microscopy image of Rac1^flox/flox; Cre system in red fluorescence, showing cell comparison results." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
 Figure 1. Comparison between Rac1flox/flox cells infected with adeno-Cre virus (left) compared to uninfected cells from the same source (right).

<ol><li>Guo, F., Debidda, M., Yang, L., Williams, D. A., Zheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+Deletion+of+Rac1+GTPase+Reveals+Its+Critical+Role+in+Actin+Stress+Fiber+Formation+and+Focal+Adhesion+Complex+Assembly%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Genetic Deletion of Rac1 GTPase Reveals Its Critical Role in Actin Stress Fiber Formation and Focal Adhesion Complex Assembly.</a> J. Biol. Chem. 281, 18652-18659 (2006).</li>
<li>Glogauer, M., Marchal, C. C., Zhu, F., Worku, A., Clausen, B. E., Foerster, I., Marks, P., GP, D. owney, Dinauer, M., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rac1+deletion+in+mouse+neutrophils+has+selective+effects+on+neutrophil+functions%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.</a> J. Immunol. 170, 5652-5657 (2003).</li>
<li>Prost, S., Sheahan, S., Rannie, D., Harrison, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenovirus-mediated+Cre+deletion+of+floxed+sequences+in+primary+mouse+cells+is+an+efficient+alternative+for+studies+of+gene+deletion%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion.</a> Nucleic Acids Res. 29, E80-E80 (2001).</li>
<li>Rijnkels, M., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenovirus-Cre-mediated+recombination+in+mammary+epithelial+early+progenitor+cells%5BTitle%5D)+AND+%22J.+Cell.+Sci%22%5BJournal%5D)">Adenovirus-Cre-mediated recombination in mammary epithelial early progenitor cells.</a> J. Cell. Sci. 114, 3147-3153 (2001).</li>
<li>Vidali, L., Chen, F., Cicchetti, G., Ohta, Y., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rac1-null+mouse+embryonic+fibroblasts+are+motile+and+respond+to+platelet-derived+growth+factor%5BTitle%5D)+AND+%22Mol.+Biol.+Cell%22%5BJournal%5D)">Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor.</a> Mol. Biol. Cell. 17, 2377-2390 (2006).</li>
<li>Wang, H., Xie, H., Zhang, H., Das, S. K., Dey, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditional+gene+recombination+by+adenovirus-driven+Cre+in+the+mouse+uterus%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">Conditional gene recombination by adenovirus-driven Cre in the mouse uterus.</a> Genesis. 44, 51-56 (2006).</li>
</ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Canadian Council on Animal Care.

The accompanying video exemplifies how a recombinant virus can be used to excise a specific gene in vitro using Cre recombinase. The development of this protocol however did reveal some particular points worth addressing.
<ul>
<li>	Proper safety procedures should always be employed when working with adenoviruses. A lab coat and gloves should be worn at all times while working with the virus. Media and plastic ware should be treated with 10% bleach for at least 30 minutes, and plastic ware should be autoclaved thereafter.</li>
<li>	Avoid repeated freeze-thawing of the virus stock as this can affect virus titer and thus reduce transduction efficiency. Small volumes of virus stock should initially be aliquoted into separate tubes.</li>
<li>	MOIs ranging from 50-500 were tested during method development and efficient transduction rates were observed in most cases, without negative effects on cell viability.</li>
<li>	Adeno-GFP was also used during method development to quickly screen transduction efficiencies. This control (or an empty vector) should be conducted during the experiment to ensure that viral infection alone does not result in cellular changes. Similarly, if excising the floxed gene of interest in your cells does not result in any cellular changes (i.e. changes in morphology or viability), then infecting the same cell type with adeno-GFP is an important control to use to demonstrate that virus transduction is occurring.</li>
</ul>
Beyond the examination of signalling cascades in culture, the adeno-Cre approach has also been employed in vivo to conditionally remove genes from experimental animals3,4, and to label specific populations of cells within an organism6. The adenovirus system can also be adapted to introduce genes other than Cre recombinase into host cells. Two principal advantages of using this delivery vector are its high transduction and gene delivery efficiency, and lack of integration with host DNA. However, the generation of novel recombinant adenoviruses can be labour intensive. The protocol described here therefore harnesses the power of the adenoviral system by exploiting the previously developed adeno-Cre virus, and coupling that with our floxed allele of interest, Rac1. Through this conditional knock out experiment, we have confirmed that transduction using the adeno-Cre virus of MEFs carrying a floxed Rac1 allele does indeed cause excision of Rac1 leading to the changes in cellular morphology characteristic of Rac1-deficient cells2,5.

<table><tbody><tr><td>Ad-CMV-Cre virus</td><td>Vector Biolabs</td><td>1045</td><td></td></tr><tr><td>DME High glucose media 500mL</td><td>Fisher Scientific</td><td>SH3002201</td><td></td></tr><tr><td>FBS Canadian origin 500mL</td><td>VWR international</td><td>CAA15-701</td><td></td></tr><tr><td>PEN-STREP 1X solution 100mL</td><td>Fisher Scientific</td><td>SV30010</td><td></td></tr><tr><td>Texas Red-X phalloidin</td><td>Invitrogen</td><td>T7471</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T4549</td><td></td></tr></tbody></table>

adenovirus-mediated genetic removal of signaling molecules in cultured primary mouse embryonic fibroblasts

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

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.

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Learn adenoviral Cre delivery for conditional gene knockout in primary mouse embryonic fibroblasts. This method achieves nearly 100% transgene efficiency to study Rac1 function in actin dynamics.

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

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