obtain frozen mouse embryonic fibroblasts ( mefs ) ( generated previously from individual embryo primary cultures ) from liquid nitrogen . thaw cells by immersing vial in 37c water with swirling until contents are completely thawed . add 9ml of dmem that has been supplemented with 10% fetal bovine serum and 1% penicillin - streptomycin to a 10 cm cell culture plate . add the thawed cell suspension drop - wise to the plate , rock the plate gently to disperse cells and place plate in a 37c , 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 .   when cells have grown to essentially 100% confluency ( ie : the next day ) , aspirate off the media and wash cells with 5ml of sterile pbs . remove pbs and add 1ml of trypsin containing 10 mm edta and place plate in incubator for approximately 5 minutes to allow cells to detach from plate . 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 37c incubator overnight . remove cells from incubator , wash and trypsinize as before , except this time add 5 - 6ml media back to the trypsinized cells instead of 9ml . pipette cells thoroughly to ensure complete dissociation into a single cell suspension , and remove 15l to combine with 15l of trypan blue in a separate microfuge tube .    mix this cell/ trypan blue suspension by pipetting up and down several times , and add 15l to the hemocytometer .    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 l of suspension :    through protocol optimization , we have determined that plating 30,000 cells results in appropriate cell density for visualization at the conclusion of the experiment .   to calculate the volume of cell suspension needed for 30,000 cells , use the following equation :    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 . 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 . 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 . 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 : 

   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 37c incubator overnight . 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 .   in the case of the rac1 cells exemplified in this experiment , morphological changes occurred in the cells at approximately 72 hours post transduction . 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 rac1 cells that were exposed to adeno - cre virus compared to the same cells not exposed to virus . comparison between rac1 cells infected with adeno - cre virus ( left ) compared to uninfected cells from the same source ( right ) . obtain frozen mouse embryonic fibroblasts ( mefs ) ( generated previously from individual embryo primary cultures ) from liquid nitrogen . thaw cells by immersing vial in 37c water with swirling until contents are completely thawed . add 9ml of dmem that has been supplemented with 10% fetal bovine serum and 1% penicillin - streptomycin to a 10 cm cell culture plate . add the thawed cell suspension drop - wise to the plate , rock the plate gently to disperse cells and place plate in a 37c , 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 . when cells have grown to essentially 100% confluency ( ie : the next day ) , aspirate off the media and wash cells with 5ml of sterile pbs . remove pbs and add 1ml of trypsin containing 10 mm edta and place plate in incubator for approximately 5 minutes to allow cells to detach from plate . 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 37c incubator overnight . remove cells from incubator , wash and trypsinize as before , except this time add 5 - 6ml media back to the trypsinized cells instead of 9ml . pipette cells thoroughly to ensure complete dissociation into a single cell suspension , and remove 15l to combine with 15l of trypan blue in a separate microfuge tube .    mix this cell/ trypan blue suspension by pipetting up and down several times , and add 15l to the hemocytometer .    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 l of suspension :    through protocol optimization , we have determined that plating 30,000 cells results in appropriate cell density for visualization at the conclusion of the experiment .   to calculate the volume of cell suspension needed for 30,000 cells , use the following equation : 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 . 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 . 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 . 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 : 

   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 37c incubator overnight .    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 .   in the case of the rac1 cells exemplified in this experiment , morphological changes occurred in the cells at approximately 72 hours post transduction . 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 rac1 cells that were exposed to adeno - cre virus compared to the same cells not exposed to virus . comparison between rac1 cells infected with adeno - cre virus ( left ) compared to uninfected cells from the same source ( right ) . the accompanying video exemplifies how a recombinant virus can be used to excise a specific gene in vitro using cre recombinase . 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 . avoid repeated freeze - thawing of the virus stock as this can affect virus titer and thus reduce transduction efficiency . 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 . 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 . beyond the examination of signalling cascades in culture , the adeno - cre approach has also been employed in vivo to conditionally remove genes from experimental animals , and to label specific populations of cells within an organism . 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 cells . experiments on animals were performed in accordance with the guidelines and regulations set forth by the canadian council on animal care .