Name: hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice
Uploaded: 2016-07-09T13:00:00.000+00:00
Duration: 7 min 30 s
Description: Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

NIH R01MH084894 participated in the funding of this study. We would like to thank Drs. Yasmin Hurd and Scott Russo at Mount Sinai School of Medicine for the donation of mice and the use of their surgery and behavior facilities during the filming of this work.

NOTE: All procedures for animal breeding and cares were conducted according to the Institutional Animal Care and Use Committee&#160;(IACUC) regulation of Icahn School of Medicine at Mount Sinai. Be sure to use sterile gloves throughout the procedure.&#160;
1. Drug and Virus Preparation
<ol>
	<li>Drug Preparation
	<ol>
		<li>Prepare 15.0 ml ketamine/xylazine anesthetic by dissolving 1.35 ml of 100 mg/ml Ketamine and 0.75 ml of 20 mg/ml xylazine in 12.9 ml of 0.9% Saline solution. Thoroughly mix solution.</li>
	</ol>
	</li>
	<li>Virus Preparation
	<ol>
		<li>Clone the mGlu2 and mGlu2&#916;TM4N constructs into a bicistronic herpes simplex virus (HSV) vector following standard protocols previously described6. Package the viral particles as previously described6,13,14. Substitution of residues Ala-6774.40, Ala-6814.44 and Ala6854.48 in mGlu2 for Ser6864.40, Phe6904.44 and Gly-6944.48 in mGlu3 (HA-mGlu2&#916;TM4N) have been described previously6. 
		NOTE: It was previously demonstrated that the chimeric construct HA-mGlu2&#916;TM4N is expressed at the plasma membrane with intact G protein-dependent signaling6.</li>
		<li>Store viral vectors in -80 &#176;C when not in use. Thaw viral vector on ice, and then aliquot into 10 &#956;l aliquots. For the surgical procedure, keep on ice.</li>
	</ol>
	</li>
</ol>
2. Surgery
<ol>
	<li>Surgery Preparation
	<ol>
		<li>Weigh mouse and inject mouse with appropriate dose of ketamine/xylazine cocktail (for details, see 1.1.1).</li>
		<li>Check mouse to see if properly anesthetized, squeeze foot and tail for pain response, if unable to elicit a response, the mouse is properly anesthetized.</li>
		<li>Shave mouse head from the base of the skull to the tip of the nose using clippers. Apply ophthalmic gel to the mouse afterward to prevent blindness of the mouse.</li>
		<li>Load each syringe onto the stereotaxic frame. Then tilt perpendicular portion of each arm of the stereotaxic frame so that they are 10 degrees away from the normal. Ensure that the arms are tilted, such that the needles are facing each other.</li>
		<li>Clean each syringe by filling the needle with 70% ethanol. Fill the needle at least three times to ensure that the syringe is clean.</li>
		<li>Once the needle has been cleaned, flush the needle by filling the needle with double distilled H2O. Once flushed, fill each needle with 1.3 &#181;l of double distilled H2O. Twist the plunger of the syringe to release 0.3 &#181;l of double distilled H2O. If water beads at the tip of the needle, carefully wipe away water. If nothing comes out of the syringe, push the plunger completely down and then repeat cleaning of syringe.</li>
		<li>After filling with water, then pull up the syringe filling the syringe with 0.5 &#181;l of air.</li>
		<li>Once the air and water are in the syringe, carefully fill the syringe with 1.3 &#181;l of virus solution. At this point ensure that the total volume in the syringe is 2.8 &#181;l. Again twist the tip of the syringe to release 0.3 &#181;l of virus. If liquid beads at the tip of the needle, carefully wipe away liquid. If nothing comes out of the syringe, push the plunger completely down and then repeat cleaning of syringe.</li>
	</ol>
	</li>
	<li>Surgery
	<ol>
		<li>Attach the mouse to the stereotaxic frame, making sure to adjust the stereotaxic frame so that the skull is level and flat. Apply povodine-iodine to the exposed scalp. Using a scalpel, make a sagittal incision along the midline of the skull within the exposed shaved area. Then attach the buret clamps to the skin at the incision site to make sure that the skull remains exposed.</li>
		<li>Use H2O2 to dissolve away the periosteum to expose the sutures of the skull. Now that the bregma and sutures are visible, be sure to adjust the stereotaxic frame to make sure that the skull is level.</li>
		<li>Align the needle tips of the syringes with the bregma and record the coordinates of the bregma. Calculate the coordinates of the where the needles are going to be inserted.
		<ol>
			<li>For the Rostral-Cauldal (R-C) plane, add 1.6 mm to the recorded R-C bregma coordinates (+1.6 from Bregma). For the Dorsal-Ventral (D-V) plane, subtract 2.4 mm from the recorded D-V bregma coordinates (-2.4 from Bregma).</li>
			<li>Finally, for the Medial Lateral (M-L) plane, add 2.6 mm to the recorded M-L bregma coordinates (+2.6 from Bregma). For all coordinates be sure to record both left and right coordinates, as this is a bilateral injection.</li>
		</ol>
		</li>
		<li>Bring the needles to the desired coordinates. Mark the places of where the needles are going to be inserted and with a drill, drill the marked areas.</li>
		<li>With a cotton tip applicator wipe away any excess blood or bone fragment.</li>
		<li>Bring the needles to the skull where the tips of the needles are touching the surface of the brain. Then lower the needles to the desired coordinates slowly lowering them.</li>
		<li>Once the needles are at the desired coordinates, slowly inject the contents of the syringe by twisting the plunger of the needle 0.1 &#181;l per minute over the course of 5 min&#160;(in total 0.5 &#181;l).</li>
		<li>Once the injection has been made leave the syringe in cortex for another 5 min.</li>
	</ol>
	</li>
	<li>Closing Up/Care
	<ol>
		<li>Remove the needles from the mouse cortex steadily and slowly. Then remove the mouse from the stereotaxic frame.</li>
		<li>Apply cyanoacrylate (dermal adhesive) to the base flaps of skin from the incision and then with forceps grab the flaps of skin and place them together.</li>
		<li>Allow the cyanoacrylate to dry. Place the mouse in cage over a heating pad (heating pad is optional if the surgical suite is kept under RT of 37 &#176;C - otherwise not necessary). Be sure to place the mouse on a paper towel to make sure that bedding does not adhere to surgical site.</li>
		<li>Depending on the length of the surgical procedure, ensure that the mice is out of anesthesia within 30 - 60 min&#160;after procedure.&#160;Following surgery, the animal is placed in its own cage and monitored until it becomes conscious before returning to its room to recover. No analgesics are used post-operatively, because they may alter the results of our experiments by modifying some of the brain biochemical pathways. However, animals are monitored until they recover from anesthesia on the day of surgery, and daily post-operation for signs of infection and evaluation of pain/distress.</li>
	</ol>
	</li>
</ol>
3. Head Twitch Response Experiment
<ol>
	<li>Set-up
	<ol>
		<li>Carry out all behavioral testing between 10:00 AM and 2:00 PM, 2 - 3 days after stereotactic injection of viral particles.</li>
		<li>Dissolve (&#177;)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) into a 0.9% saline solution to 2.0 mg/kg. Also prepare a 0.9% saline solution.</li>
		<li>Prepare a home cage (28 x 18 x 15 cm) without any bedding and using a tri-pod, adjust a camera so that the view of the video camera is directly above the home cage.</li>
		<li>Habituate the mice to the room for at least 4 hr prior to the beginning of the experiment.</li>
		<li>Set up a camcorder to record the head twitch.</li>
	</ol>
	</li>
	<li>Experiment
	<ol>
		<li>Position the camera so that it is directly over a home cage. Calibrate the camera so that the entire home cage is in the field of view.</li>
		<li>Weigh mouse and inject mouse intraperitoneally with appropriate dose of either 0.9% saline or DOI (0.01 ml/g). NOTE: If a mouse weighs 25 grams, administer dose to a total volume of 0.25 ml.</li>
		<li>Place each mouse back in their home cage for 10 min. After 10 min, place mouse into the center of the empty home cage and validate that there are no blind spots in the field of view of the camera. Press record on the camcorder. Leave the room. 
		NOTE: Mouse movements and various behavioral responses therein (head twitch, ear scratch, etc. Please refer to supplemental data table 1 of Gonzalez-Maeso et al. 2007 for full list of behavioral responses induced by DOI.)3, will be recorded for 30 min. Therefore, it is important there are no blind spots in the field of view recorded.</li>
		<li>After 30 min&#160;stop recording on the camcorder and place mouse back in original home cage. Repeat this process for each mouse.</li>
	</ol>
	</li>
	<li>Review
	<ol>
		<li>Have each referee review the tapes blind to the experimental conditions of mouse (i.e., drugs used during head twitch experiment or virus used during intracranial injection)). Manually record every head twitch throughout the video. 
		NOTE: Head-twitch is defined as a rapid shaking head movement conducted by a mouse (supplemental video).</li>
		<li>For each mouse, average the final HTR response from the three totals of the blind referees. Then group these values by experimental condition and carry out statistical analysis (i.e., t-test or ANOVA).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Together with previous findings in mGlu2-KO mice5, the results with mGlu2 and mGlu2/mGlu3 chimeric constructs that do not form the 5-HT2A-mGlu2 receptor complex in cultured cells suggest that the 5-HT2A -mGlu2 heteromeric receptor complex in mouse frontal cortex is needed to induce head-twitch behavior by LSD-like hallucinogenic 5-HT2A receptor agonists. A limitation of this method is that it does not measure close molecular proximity at a subcellular level in native tissue. In addition, there are various critical points to be noted. Because the mice are injected with a HSV viral vector, the time frame that the experiments to be performed are 2 - 4 days after injection. The location and expression of the viral vectors should be verified with immunofluorescence staining of sectioned brain no more than 4 days after the initial injection. Mice in which the coordinates do not match or do not express the viral vector should be excluded from the experimental data as they do not express the mGlu2 or mGlu2&#916;TM4N. Care after stereotactic surgery is also crucial, as improper closing of the head wound can lead to infection which can cause issues in both the in vivo experiments and the immunofluorescence staining. Lastly, after stereotactic injection any behavioral paradigm (open field, alternating t-test, etc.) can be used as long as it is within the 2 - 4 after injection of viral particles. Again, coordinates and expression should be confirmed by immunofluorescence.
The concept that GPCRs function as homo- and/or heteromers in living cells is now well established 16-18. However, despite some progress, more studies are needed to define the precise role(s) of GPCR heteromeric complexes in whole animal models 19. Approaches such as BRET and/or FRET imaging to investigate protein-protein physical proximity within deep tissues of small animal models may provide means to enhance the understanding of the functional role of GPCR heteromers in living subjects20. Using HSV-mediated expression of GPCRs either native or with modified transmembrane proteins, provide an in vivo model to evaluate the function and interaction of GPCRs.
Further studies in rodent models are also needed to examine the stability and life- time (formation and dissociation) of GPCR heteromers21-23, structural rearrangements between their components24, and potential G protein coupling after receptor internalization25,26. Given the fundamental role of GPCRs in cell signaling and function, it seems likely that this area might lead to interesting basic and translational studies.
Although further investigation is required to quantitatively characterize the ultrastructural co-localization of both receptors in human and mouse CNS, together with previous studies that convincingly demonstrate the electrophysiological, cellular, and behavioral responses induced by hallucinogens in mouse models are intrinsic to 5-HT2A receptor-expressing cortical pyramidal neurons3,27,28, the findings obtained using the HSV-mediated expression approach described here suggest that heteromeric formation between 5-HT2A and mGlu2 receptors in mouse frontal cortex is needed for the head-twitch psychosis-like behavior induced by the hallucinogenic 5-HT2A receptor agonist DOI.
HSV-mediated expression of mGlu2, but not mGlu2&#916;TM4N, in frontal cortical neurons of mGlu2-KO mice rescues the head-twitch behavior induced by the hallucinogenic 5-HT2A receptor agonist DOI. This translational tool might be advantageous for preclinical studies to evaluate behavioral phenotypes of GPCR heteromers.

Hallucinogens, such as LSD, psilocybin and mescaline cause significant changes in human consciousness, cognition and emotion7-9. Inactivation of serotonin 5-HT2A receptor signaling by either genetic or pharmacological approaches causes markedly attenuated behavioral responses to hallucinogens in both rodent models3,10 and humans11. Although hallucinogens bind other receptor subtypes8, the 5-HT2A receptor is considered as necessary for the unique behavioral activity of these chemicals.
Group II metabotropic glutamate receptors (i.e., mGlu2 and mGlu3) have been the target of considerable attention regarding the molecular mechanism of hallucinogens and their integral role underlying psychosis12. Previously, it has been demonstrated that mice with no expression of mGlu2 protein (mGlu2-KO mice) are insensitive to the cellular and behavioral effects of hallucinogens5. It has also been suggested that the 5-HT2A and the mGlu2 receptors form a specific heteromeric complex through which serotonin and glutamate ligands modulate the pattern of G protein coupling in living cells 1,2.
Structurally, transmembrane (TM) domains 4 and 5 of mGlu2 play a fundamental role in heteromeric formation with the 5-HT2A receptor5. Additionally, further investigation demonstrated that three residues located at the intracellular end of TM4 of mGlu2 are necessary to form the 5-HT2A-mGlu2 receptor heterocomplex in living cells6.
Based on these findings observed in heterologous expression systems, here we describe the use of HSV-mediated expression of wild-type mGlu2 and mGlu2/mGlu3 chimeric constructs in the frontal cortex of mGlu2-KO mice to test whether heteromeric formation between 5-HT2A and mGlu2 is necessary for the head-twitch behavior induced by hallucinogenic 5-HT2A receptor agonists.

<table><tbody><tr><td>mGlu2 bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></td><td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td></tr><tr><td>mGlu2&amp;Delta;TM4N bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></td><td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td></tr><tr><td>GFP bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></td><td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td></tr><tr><td>Xylazine&amp;nbsp;</td><td>Lloyd</td><td>List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10</td><td>1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>Vedco</td><td>KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06</td><td>1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution</td></tr><tr><td>Ophthalmic gel</td><td>Fisher Scientific</td><td>NC0550805</td><td></td></tr><tr><td>Burret clips</td><td>Fisher Scientific</td><td>NC9268369</td><td></td></tr><tr><td>Feather surgical blade</td><td>Fisher Scientific</td><td>NC9032736</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Fisher Scientific</td><td>19-898-919&amp;nbsp;</td><td></td></tr><tr><td>Hamilton syringe</td><td>Fisher Scientific</td><td>14815203</td><td></td></tr><tr><td>Hamilton&amp;trade; Small Hub Removable Needles (33 Ga)</td><td>Fisher Scientific</td><td>14816206</td><td></td></tr><tr><td>Cordless Micro Drill</td><td>Fisher Scientific</td><td>NC9089241</td><td></td></tr><tr><td>Dermabond Dermal Adhesive</td><td>Fisher Scientific</td><td>NC0690470</td><td></td></tr><tr><td>(&amp;plusmn;)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)</td><td>Sigma-Aldrich</td><td>42203-78-1</td><td>Dissolved in 0.9% saline solution to the concentration of 2.0 mg/kg</td></tr></tbody></table>

hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

Previous findings demonstrate that the head-twitch murine behavioral response is reliably and robustly elicited by hallucinogens, and it is absent in 5-HT2A-KO mice3. Furthermore, it has been shown that the head-twitch response elicited by the hallucinogenic 5-HT2A agonists DOI and LSD was significantly decreased in mGlu2-KO mice5. However, although previous findings convincingly demonstrate that 5-HT2A and mGlu2 are assembled as a heteromeric complex in vitro in transfected cells1,2,15, whether this structural arrangement behaves as such in living mice remained unsolved. To fully understand the role of the 5-HT2A-mGlu2 receptor heterocomplex in the psychoactive-like effects induced by hallucinogenic 5-HT2A receptor agonists, expression of either mGlu2 or mGlu2&#916;TM4N in frontal cortex of mGlu2-KO mice to examine whether this manipulation regulates behavior.
Mice received intra-frontal cortical injections of bicistronic HSV expressing green fluorescent protein (GFP) and either mGlu2 or mGlu2&#916;TM4N, or GFP alone. First, it was confirmed that the virus over-expresses mGlu2 or mGlu2&#916;TM4N in mouse frontal cortex (Figures 1A and 1B). As previously demonstrated5, head-twitch behavior induced by DOI was absent in mGlu2-KO mice injected with the empty vector HSV-GFP. Notably, the head-twitch response induced by the hallucinogenic 5-HT2A agonist DOI was rescued in mGlu2-KO mice over-expressing mGlu2, but not mGlu2&#916;TM4N, in frontal cortex as compared to that seen in animals expressing GFP (Figure&#160;1C). Together, these findings suggest that the 5-HT2A-mGlu2 receptor complex in frontal cortex is critical for regulating psychosis-like states.
<img alt="Figure 1" src="/files/ftp_upload/53717/53717fig1.jpg" /> 
Figure 1. Expression of mGlu2 as a Receptor Heterocomplex. Expression of mGlu2 as a receptor with 5-HT2A is necessary for psychosis-like behavior induced by hallucinogenic drugs. (A) Representative image of HSV-mediated transgene expression in frontal cortex. HSV-mGlu2, which also expresses GFP, was injected into frontal cortex, and GFP expression was revealed by immunocytochemistry, scale bar, 200-um. (B) HSV-mediated transgene expression in mouse frontal cortex of mGlu2-KO mice, and anti-mGlu2 reactivity was measured by Western Blotting. Specificity of the primary antibody against the mGlu2 receptor has previously been confirmed in knockout mice6. Metabotropic glutamate receptors are GPCRs that form covalently linked homodimers. We measured immunoreactivity of mGlu2 as a monomer (100 kDa)6. (C) Viral-mediated expression of mGlu2, but not mGlu2&#916;TM4N, in frontal cortex of mGlu2- KO mice significantly rescues the head-twitch response induced by the hallucinogenic 5-HT2A agonist DOI (n = 4 per group). ***p &#60;0.001; n.s, not significant; Bonferroni&#39;s post hoc test of one-way ANOVA. Error bars represent S.E.M. Figure was modified from Moreno et al (2012)6. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53717/53717fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53717/HeadTwitchResponse.wmv" target="_blank">Supplemental Video 1. Head Twitch Response. (Right click to download).</a> CD-1 WT mice were injected with 2.0 mg/kg DOI and placed in a cage (wall blacked out between the two cages) to elicit head-twitch response (behavior elicited after *).

Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

hsv-mediated-transgene-expression-chimeric-constructs-to-study

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

7.2K Views.  Icahn School of Medicine at Mount Sinai. This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

Video: HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Genetics

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

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 ...

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. However, the time and cost associated with colony creation and maintenance are high. Recent advances in CRISPR-mediated genome editing have allowed the generation of somatic GEMMs (S-GEMMs) by directly targeting the cell/tissue/organ of interest.
The oviduct, or fallopian tube in humans, is considered the tissue-of-origin of the most common ovarian cancer, high-grade serous ovarian carcinomas (HGSCs). HGSCs initiate in the region of the fallopian tube distal to the uterus, located adjacent to the ovary, but not the proximal fallopian tube. However, traditional mouse models of HGSC target the entire oviduct, and thus do not recapitulate the human condition. We present a method of DNA, RNA, or ribonucleoprotein (RNP) solution microinjection into the oviduct lumen and in vivo electroporation to target mucosal epithelial cells in restricted regions along the oviduct. There are several advantages of this method for cancer modeling, such as 1) high adaptability in targeting the area/tissue/organ and region of electroporation, 2) high flexibility in targeted cell types (cellular pliancy) when used in combination with specific promoters for Cas9 expression, 3) high flexibility in the number of electroporated cells (relatively low frequency), 4) no specific mouse line is required (immunocompetent disease modeling), 5) high flexibility in gene mutation combination, and 6) possibility of tracking electroporated cells when used in combination with a Cre reporter line. Thus, this cost-effective method recapitulates human cancer initiation.

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

This protocol describes microinjection and in vivo electroporation for regionally restricted CRISPR-mediated genome editing in the mouse oviduct.

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. ...

Cancer Research

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

Bioengineering

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

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Rozdziały w tym wideo

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

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

Viral Tracing of Genetically Defined Neural Circuitry

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals