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&deg;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&deg;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&deg;C incubator overnight. The presence of FBS in the media will inactivate the trypsin &#8211; 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 15&mu;L to combine with 15&mu;L 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 15&mu;L 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 &mu;L of suspension: 
 <img src="/files/ftp_upload/2160/2160eq0.jpg" 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="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 3" /> 
 
 <img src="/files/ftp_upload/2160/2160eq3.jpg" 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&deg;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="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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Deletion+of+Rac1+GTPase+Reveals+Its+Critical+Role+in+Actin+Stress+Fiber+Formation+and+Focal+Adhesion+Complex+Assembly.">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. o. w. n. e. y., Dinauer, M., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1+deletion+in+mouse+neutrophils+has+selective+effects+on+neutrophil+functions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+Cre+deletion+of+floxed+sequences+in+primary+mouse+cells+is+an+efficient+alternative+for+studies+of+gene+deletion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-Cre-mediated+recombination+in+mammary+epithelial+early+progenitor+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1-null+mouse+embryonic+fibroblasts+are+motile+and+respond+to+platelet-derived+growth+factor.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+recombination+by+adenovirus-driven+Cre+in+the+mouse+uterus.">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

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.

Watch this Scientific Journal Video about Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts at JoVE.com

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

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 ...

adenovirus-mediated-genetic-removal-signaling-molecules-cultured

Nanyang Technological University

Research

JoVE Journal

Biology

25.0K Views.  University of Guelph. In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele. 

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

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Developmental Biology

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-&#946; is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-&#946;/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-&#946; displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-&#946; signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-&#946; signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Here, we present a protocol for live cell imaging of TGF-&#946;/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

Cancer Research

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based approaches. For example, although chimeric and Cre-LoxP targeting strategies have been widely used, the intrinsic limitations of these strategies (i.e., leaky promoter activity, mosaic Cre expression, etc.) have significantly restricted their application. Moreover, a complete deletion of many endogenous genes is embryonically lethal, making it impossible to study gene function in postnatal life. To address these challenges, we have made significant changes to an early genetic engineering protocol and combined a short (transgenic) version of the Rb1 gene with a lysosomal protease procathepsin B (CB), to generate a DN mouse model of Rb1 (CBRb). Due to the presence of a lysosomal protease, the entire CB-RB1 fusion protein and its interacting complex are routed for proteasome-mediated degradation. Moreover, the presence of a tetracycline inducer (rtTA) element in the transgenic construct enables an inducible and reversible regulation of the RB1 protein. The presence of a ubiquitous ROSA-CAG promoter in the CBRb mouse model makes it a useful tool to carry out transient and reversible Rb1 gene ablation and provide researchers a resource for understanding its activity in virtually any cell type where RB1 is expressed.

Inducible and Reversible Dominant-negative DN Protein Inhibition

Here we present a protocol to develop a dominant-negative inducible system, in which any protein can be conditionally inactivated by reversibly overexpressing a dominant-negative mutant version of it.

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based ...

Genetics

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Neuroscience

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Immunology and Infection

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

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

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Chapters in this video

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation