This work was supported by grants to JLRR from: Autism Speaks, Nina Ireland, Weston Havens Foundation, NIMH R01 MH081880, and NIMH R37 MH049428. PRW was supported by a fellowship from the National Science Council of Taiwan. SFS was supported by F32 (MH103003).

Ethics statement: The following procedures have been approved by our institution and animal protocol. Make sure to get approval for all procedures involving survival surgeries before beginning experiments and verify all protocols are up to date.
1. Lentivirus Preparation (Optional Step)
<ol>
	<li>Split three 10 cm plates of HEK293T cells for each lentivirus to be made, in the presence of 10 ml DMEM/10% FBS, and grow to ~60-70% confluence. Grow cells in an incubator at 37 &#176;C with 5% CO2.</li>
	<li>Transfect the following DNA plasmids (10 &#181;g total) into each plate using any transfection reagent: 6.4 &#181;g CAG-Flex-GFP lentiviral vector (DNA vector sequence provided in Table 1), 1.2 &#181;g pMD2.G (encodes VSV-g), 1.2 &#181;g pRSV-Rev, and 1.2 &#181;g pMDLg/pRRE. The three lentiviral packaging vectors are commercially available (Table 
	Note: This particular approach utilizes 3rd generation lentiviral vectors but 2nd generation vectors and associated plasmids will also work.</li>
	<li>Prepare a waste container containing a 10% bleach solution within a BSL2 certified hood to inactivate any solutions or devices that contacted or contain lentivirus. Next, completely remove the media 4-6 hr&#160;after transfection into the bleach solution, replace with the same volume of new media, and allow the cells to grow.</li>
	<li>After 4 days, collect media into 50 ml conical tubes and centrifuge at 1,000 x g for 15 min to pellet any cell debris. Next, filter the supernatant through a 0.45 &#181;m membrane. Any low protein binding filter, like PVDF, works well. Caution: place both solid and liquid viral waste into the bleach solution.</li>
	<li>Load 30 ml filtered supernatant into ultracentrifuge tubes and into an ultracentrifuge. Centrifuge at 100,000 x g for 2.5 hr at 4 &#176;C. Open the centrifuge tubes in a BSL2 hood and remove supernatant into 10% Bleach. Add ~100 &#181;l of PBS to the pellet, which yields titers of 1x10^7-8 infectious units/ml. Aliquot the following day and store at -80 &#186;C. Lentivirus aliquots are stable at -80 &#176;C for at least six months.</li>
	<li>Important consideration: Many institutions now have viral cores. Acquire concentrated virus with a minimum titer of 1x107 infectious units/ml, for optimal results.</li>
</ol>
2. Donor Mice for MGE Dissection
<ol>
	<li>First, set up a timed mating between a Cre-expressing line of interest and either a wild type (WT) mouse or a mouse that will express a reporter after Cre-mediated recombination. 
	Note: Many Cre-driver lines are transgenics and are maintained and bred in the hemizygous state. Thus, only half of the embryos will contain the Cre transgene. DNA can be quickly prepared from the embryos for a short PCR to detect the Cre allele. The Cre+ embryos can then be harvested so that only the MGE tissue of the desired genotype is used. Alternatively, a Cre-dependent reporter may be used, to select the embryos for MGE dissection and transplantation.</li>
	<li>Calculate which day will correspond to E13.5. If animals were paired the evening before, the day of the plug is 0.5. Order timed-pregnant mice or a WT female with P1 pups in order to time having postnatal (P)1 mice on the day the donor tissue will be E13.5. Alternatively, set up these matings in house one week prior to pairing the donor animals. 
	Note: The former strategy of generating both E13.5 and P1 embryos/pups for the same day is often difficult to do reliably.</li>
</ol>
3. Preparation of Media, Tools and Equipment.
<ol>
	<li>Prepare the reagents and tools for MGE cell preparation (Table 2).
	<ol>
		<li>Prepare a clean surface and place clean forceps, scissors and either a microsurgical knife or fine forceps (for precise tissue dissection) on the surface.</li>
		<li>Prepare Hank&#39;s balanced salt solution (HBSS), for dissection, and Dulbeco&#39;s modified eagle&#39;s medium (DMEM) with 10% fetal bovine surem (FBS), for transduction.</li>
		<li>Preincubate 20-30 ml DMEM/10% FBS in a 37 &#176;C tissue culture incubator for about 1 hr. The lid should be loosened to allow for gas exchange and pH equilibration of the media. 
		Note: The media preincubation can be prepared just before starting the dissection.</li>
	</ol>
	</li>
</ol>
4. MGE Cell Preparation
<ol>
	<li>Sacrifice the pregnant mouse according to an approved animal protocol and remove the embryos into a 10 cm petri dish containing ice-cold HBSS.</li>
	<li>Remove the brain from each embryo using a dissecting scope (do one at a time, leaving the rest of the embryos in ice-cold HBSS) and then proceed to cut out the MGE tissue. 
	Note: Detailed below, and in Figure 1, are key steps showing how to remove the MGE. In addition, Figure 2 is a movie showing the entire dissection procedure.
	<ol>
		<li>Position the brain with ventral side facing up (Figures 1A, A&#39;). Note: it is also okay to have the dorsal aspect facing up. Cut the brain in half along the saggital plane to generate two pieces. An example hemisected brain is shown (Figures 1B, B&#39;), notice that dorsal (the overlying cortex) and ventral aspects of the brain cover and surround the ganglionic eminence (GE) tissue.</li>
		<li>Next, with a dominant hand, hold either very fine forceps or a stabknife (Table 2), while immobilizing the hemisphere with forceps held by a non-dominant hand. (The hemisphere&#39;s medial aspect should be facing up). Unfold the dorsal and ventral tissue with forceps and a stab knife to reveal the underlying GE tissue (Figure&#160;1C and diagrammed in Figure&#160;1C&#39;). 
		Note: Removing some media from the dissection petri dish can make it easier to stabilize the tissue while performing the MGE dissection.</li>
		<li>Make straight cuts with the stabknife or fine forceps to separate the CGE, LGE and septal tissues (see example cuts denoted by red dashed lines, Figures 1D and D&#39;). These cuts can be made in any order. 
		Note: Imagine the MGE as a &#39;box&#39; that is cut out of the surrounding tissue. Doing so helps reproducibly make similar cuts for each dissection, thus reducing variability in tissue dissections.</li>
		<li>Finally, turn the MGE on its side and trim off the bottom (what was the lateral aspect of the brain). This removes the mantle zone of the MGE (discard in Figures 1E and 1E&#39;) from the rest of the MGE. The remaining aspect of MGE contains primarily ventricular zone and subventricular zone portions of the MGE, which contains nearly all the MGE progenitor cells. Proceed with this tissue.</li>
	</ol>
	</li>
	<li>Important consideration: The addition of silicone gel to the bottom of a petri dish can serve as an excellent dissection surface as it protects the delicate tips of forceps and provides a soft surface for pinning tissue with forceps.</li>
</ol>
4.3&#160;Additional strategies for MGE Dissection.
<ol>
	<li>Cut the skin of the embryo using forceps like scissors to quickly remove the brain. As the embryo lays on its side, hold the tissue at the base of the head with the forceps used to anchor the tissue. First, cut anterior ventral to the brain and posterior ventral to the brain, and then connect the two cuts along the side of the embryo. Then grab the flap of skin and pull it over the top of the brain. The brain can then be quickly dissected away from the remaining tissue.</li>
	<li>Alternatively, anchor the posterior aspect of the whole brain behind the cortex. Meanwhile, grasp each hemisphere at the most dorsal caudal aspect of the cortex and tear the cortex away from the anchored tissue in an anterior direction. In this way, both cortices can be removed and the bilateral ganglionic eminences are revealed, still attached to the rest of the tissue preserving the medial-lateral anatomy.</li>
	<li>Switch to the stab-knife to make precise cuts around the MGE on each hemisphere. Use either a separate clean set of forceps to collect the MGE or a pipette with a large tip (i.e., P1000). 
	Note: Keep the collected tissue away from all other dissected material, as some media is unavoidably transferred when the MGE is collected.</li>
	<li>Collect the MGE and put the tissue into a 1.5 ml collection tube containing DMEM/10% FBS, (a volume of 500 &#181;l&#160;is sufficient to collect the MGEs). Repeat for the other hemisphere and place in the same collection tube. Keep samples on ice until all tissue is collected.</li>
</ol>
5. Lentiviral labeling and Transplantation
<ol>
	<li>Move the tubes of MGE tissue into a BSL2 certified hood and remove the media. 
	Note: The MGE tissue is big enough to be visible to the eye and will settle to the bottom of the tube allowing for the cold media to be easily and gently removed.</li>
	<li>Add ~500 ul&#160;of DMEM/10% FBS media, that was preincubated in a 37 &#176;C tissue culture incubator. Next, add polybrene to facilitate transduction to each tube at a final concentration of 8 &#181;g/ml. Triturate the MGE tissue to create a single cell suspension, using a P1000 pipette tip. Finally, add ~15-20 &#181;l of concentrated lentivirus to each tube.</li>
	<li>Important consideration: A trituration of 10-12 times is sufficient to break up MGE tissue from a single embryo, but more triturations may be required if multiple MGEs are pooled together. Try out different trituration conditions to determine what works best.</li>
	<li>Securely close each tube, invert to mix, than place all the tubes in a 37 &#176;C incubator. Incubate the cells in lentivirus for at least 30 min and up to 1 hr. Longer times have resulted in decreased cell viability. Invert the tubes every 10 min until the time is up.</li>
	<li>After incubation with lentivirus, remove the tubes from the incubator and centrifuge at ~700 x g for 3 min to pellet the cells. In the BSL2 hood, remove the supernatant and discard into 10% bleach. Next, add 1 ml DMEM/10% FBS and triturate the pellet 2-3 times to wash, then centrifuge again at the same speed to pellet the cells. Repeat this wash-step 2-3 more times to remove excess virus. 
	Note : Perform centrifugation steps at 4 &#176;C, however, reduced viability has not been observed when short spins are performed at RT.</li>
	<li>After the final wash, remove as much media as possible and put each tube on ice. Due to residual media on the sides of the tube, ~2-3 &#181;l of media will end up covering the cell pellet by the time the transplantation procedure has begun.</li>
</ol>
6. Transplantation and Validation
<ol>
	<li>Acquire host P1 pups and prepare the procedure area by spraying down with 70% ethanol. To maintain sterility during the procedure it is recommended to perform these procedures in a dedicated clean procedure room. In addition, use either autoclaved surgical tools sterilize surfaces that the pups will be in contact with using 70% ethanol. An example of representative equipment needed to perform the transplantations and a representative procedure area is shown in Figure 3.</li>
</ol>
Note: While this procedure is an injection of small volumes, and not a surgical procedure that would require an incision, open wound or sutures, it is still recommended that a new micropipette is used for each mouse to be injected. The micropipettes are heat sterilized when pulled and beveled on a surface that was sprayed with 70% ethanol being stored in a sealed container.
<ol>
	<li>Generate micropipettes to have a diameter at the tip between 40-80 &#181;m. Following these dimensions will yield optimal results. Heat sterilize the micropipettes when pulled and beveled on a surface and spray with 70% ethanol before storing the micropipettes in a sealed container. 
	Note: Alternatively, if access to the devices needed to create these needles (Table 2) is limiting, use any other injection device. However, these may not be as well suited as the micropipettes due to different diameters.</li>
	<li>Draw up mineral oil into a 1 ml syringe, and with a 30&#160;1/2 G needle, fill the glass pipette completely with mineral oil. Next, mount the plunger onto the stereotaxic device, then attach the glass pipette to the stereotaxic device and load onto the plunger. Using the hydraulic drive, move the plunger about halfway into the glass pipette, removing mineral oil that spills out with a clean a clean paper towel.
	<ol>
		<li>Alternatively to using a syringe, submerge the back end of the pipette in mineral oil and filled by capillary action. To prevent air bubbles in the pipette, maintain positive pressure on the syringe until completely out of the glass pipette. 
		Note: Other devices can be used to successfully transplant MGE cells 14. Any device that can deliver a volume less than or near 100 nl works well for this procedure.</li>
	</ol>
	</li>
	<li>Next, use a sterile paper towel, or any absorbent material, with the corner twisted into a fine point to delicately remove excess media above the MGE cell pellet. This step concentrates the cell density so that smaller injection volumes can be achieved. Next, use a low volume (P2 is preferred) pipette to slowly draw up the cell pellet and triturate 1-2 times before moving the cell suspension onto a hydrophobic surface. The volume should be just under 1 &#181;l. Move the tip of the needle into the cell suspension and using the hydraulic drive, draw up the cell suspension into the pipette.</li>
	<li>&#160;Anaesthetize a P1 pup via hypothermia (wrap in a latex glove and put on ice for ~ 2-4 min). Check for effectiveness of anesthesia with a noxious stimulus. Pinch the skin between the toes with fine forceps: the pup should be unresponsive if pinched. Next, place the pup onto a mold that is under the injection device, then make the skin taut by pulling back the skin on the head and securing with standard lab tape. 
	Note: While hypothermia is very effective on neonatal mice and results in low mortality rate, procedures must be done quickly, as pups will begin to recover in under 10 min. In addition, 4 min on ice is an upper limit of what can be tolerated. Try to only induce hypothermia once if a longer time is required for injections. However, if a pup recovers early, first allow the pup to fully recover before inducing hypothermia again. Moreover, only induce hypothermia one more time to perform injections.&#160;&#160;Pups will recover within 5-10 min&#160;of hypothermia. This can be facilitated by placing the pups with litter and mom or by placing the pup on a warm surface to recover.</li>
	<li>Using a stereotaxic device, position the tip of the glass pipette on the surface on the pup&#39;s head, perpendicular to the surface of the head. When the pipette is in contact with the head, consider this as &#39;0&#39;. Next, push the pipette through the surface of the skin and skull into the cortex, then insert and retract the pipette ~ 2 times before stopping. For optimal targeting of cells into the neocortex, injections are performed when the micropipette is at a depth of 0.1 mm. However, this should be optimized by the user (see note below). 
	Note: While this depth reliably targets deeper neocortical layers with the devices described above, a few depths should be tested determined empirically. In addition, a range of depths at different rostro-caudal and medio-lateral position should be tested to determine what depth is optimal at each site. Also, the initial puncture should be rapid, as slow penetration into the neocortex diminishes the ability to cleanly penetrate the tissue. Try different speeds when first starting this procedure to find what works best. In addition, for testing coordinates, there is no real substitute for using labeled cells, as dyes have been unreliable to denote position.</li>
	<li>Once the needle is in position, rotate the hydraulic device to advance the plunger into the glass pipette, pushing a set volume of cell suspension into the cortex. Inject 50-70 nls per site. Repeat this procedure at 3-6 different sites at different rostral-caudal levels if the goal is to obtain a wide distribution of cells throughout the neocortex. 
	Note: Volumes of 100 nl or greater can cause lesions and lead to large clumps of injected cells that fail to efficiently migrate out of the injection site. Thus, smaller injection volumes (~70 nl or less) are optimal, over more sites if time permits.</li>
	<li>When injections are complete, remove the pup from the stage and mark it (toe clipping is a reliable way to identify the pups later). Once the pup has recovered and is able to move on its own (this occurs within minutes of removing them from the stage), put it back with the mom and litter. 
	Note: Since this is a minimally invasive procedure there are no post-surgical procedures once the pup is freely moving and warmed. However, pups should be checked not only after the procedure but also the following day to assure they have no signs of deteriorating health.</li>
	<li>Before starting the procedure on the next pup, spray down the area with 70% ethanol&#160;to maintain a sterile working area. Allow the injected pups to develop and then assess the tissue at the appropriate developmental stage(s).</li>
</ol>

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The authors declare that they have no competing financial interests.

The use of GABAergic cortical interneuron precursors from the embryonic ganglionic eminences (GEs) for cell based therapies is showing promise for many conditions 12&#8211;14. Precise molecular techniques are needed to track, and express genes of interest in specific interneuron subgroups. Here we provide a detailed protocol for labeling embryonic MGE cells with lentiviruses before transplantation and show how this technique can be used to express genes of interest in specific cortical in...

GABAergic cortical interneurons comprise ~20-30% of neurons in the mammalian neocortex, while the rest correspond to excitatory, glutamatergic principal neurons. Interneurons are highly diverse in electrophysiological properties, axon and dendrite morphology and synaptic targeting 1, and imbalances in excitatory/inhibitory tone are hypothesized to underlie some phenotypes of neurological/neuropsychiatric disorders including autism, schizophrenia and epilepsy 2. The overall goal of the protocol described herein is to provide a means to efficiently genetically modify GABAergic cortical interneuron progenitors before transplantation for in vivo analyses.
Cortical GABAergic interneurons are born in the medial and caudal ganglionic eminences (MGE and CGE, respectively) 3,4 as well as the preoptic area 5. Cortical interneuron progenitors undergo long-distance tangential migration followed by radial migration to reach their final targets. Upon arrival at their destinations, these cortical interneurons must correctly integrate into the existing neuronal network, and each unique interneuron subgroup will contribute to cortical circuitry in specific ways. Four main subgroups can be distinguished by molecular markers: MGE-derived somatostatin (SST)+ and parvalbumin (PV)+ subgroups, and CGE-derived vasoactive intestinal peptide (VIP)+ and Reelin+;SST- subgroups 6. Different cortical interneuron subgroups are born over different times during embryonic development in the MGE and CGE 7, 8. These and other cortical GABAergic interneuron markers have been used to generate specific Cre-driver lines for many of these subgroups 9-11.
The transplantation of MGE progenitors has emerged as a potential cell-based therapy to treat disorders that may be caused by imbalances in excitation/inhibition 12&#8211;24. These therapeutic benefits may be due in part to the unique ability of MGE progenitors (to disperse, differentiate and integrate into a host brain), or potentially because many peri-somatic inhibitory PV+ cells are derived from the MGE. MGE cells can also be quickly and efficiently transduced with lentiviruses before transplantation 15, allowing cells that are genetically modified in vitro to be studied in vivo. The rationale for developing this approach was to overcome roadblocks in studying GABAergic cortical interneuron development and maturation. In particular, MGE transplantation allowed researchers to study the development of mutant cells in vivo, when the mutant mouse would have otherwise died at an early time point. Moreover, by introducing genes of interest before transplantation, the effects of specific genes on a mutant phenotype could be assessed in an efficient manner.
Here, we provide a detailed protocol to transduce MGE cells with lentiviruses prior to transplantation. In addition, we show how this technique can be adapted to express a gene of interest in specific interneuron subgroups from a heterogeneous group of cortical interneuron precursors, using a combination of Cre-dependent expression lentiviruses and available Cre-driver mouse lines. Moreover, this protocol introduces techniques and a platform for researchers to genetically modify GABAergic cortical interneuron precursors for in vivo studies in a unique way. One advantage of this technique over other current approaches is that the transplanted MGE cells will disperse away from the injection site. Also, unlike focal viral injections, after MGE cells disperse their morphology is easier to assess. This approach can be used to study the effect of introducing genes of interest into wild type or mutant cells, introducing a cell type specific reporter to assess morphology, or potentially to study the effect of disease alleles in vivo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and equipment for MGE cell transplantation</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>REF 309602</td>
			<td colspan="2">BD, Becton Dickinson and company</td>
		</tr>
		<tr>
			<td>301/2 gauge needle</td>
			<td>305106</td>
			<td colspan="2">BD, Becton Dickinson and company</td>
		</tr>
		<tr>
			<td>Precision bore to deliver 5 &#181;l (comes with plunger)</td>
			<td>5-000-1005</td>
			<td colspan="2">Drummond scientific company</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>PM-999</td>
			<td colspan="2">Polysciences, Inc.</td>
		</tr>
		<tr>
			<td>Stereo microscope with boomstand **</td>
			<td>MZ6</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital just for mice stereotaxic instrument **</td>
			<td>51725D</td>
			<td colspan="2">Stoelting company</td>
		</tr>
		<tr>
			<td>Single-axis oil hydraulic fine micromanipulator **</td>
			<td>MO-10</td>
			<td>Narishige</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond coated rotary beveler</td>
			<td>n.a.</td>
			<td colspan="2">made in house</td>
		</tr>
		<tr>
			<td>Needle pipette puller</td>
			<td>730</td>
			<td colspan="2">Kopf instruments</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>n.a.</td>
			<td colspan="2">Any drug store or pharmacy</td>
		</tr>
		<tr>
			<td colspan="2">Any pipette and tips that can reliaby measure 1 &#181;l of volume</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">** These are the particular models we use, but many other setups should work</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Optional Reagents (for making Lentiviruses)</td>
		</tr>
		<tr>
			<td>25 mm, 0.45 &#181;m filter</td>
			<td>09-719B</td>
			<td colspan="2">Fisher Scientific</td>
		</tr>
		<tr>
			<td>Ultra-clear centrifuge tubes (25 x 89 mm)</td>
			<td>344058</td>
			<td colspan="2">Beckman Coulter&#174;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (1.5 ml size)</td>
			<td>11668019</td>
			<td colspan="2">Invitrogen</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other commercially available supplies</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE plasmid encoding gag/pol</td>
			<td>12251</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-Rev plasmid encoding Rev</td>
			<td>12253</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G plasmid encoding VSVG</td>
			<td>12259</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-Flex-GFP</td>
			<td>28304</td>
			<td>Addgene</td>
			<td></td>
		</tr>
	</tbody>
</table>

viral-mediated labeling and transplantation of medial ganglionic eminence (mge) cells for in vivo studies

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically diverse. Inroads have been made in understanding the roles of distinct cortical interneuron subgroups, however, there are still many mechanisms to be worked out that may contribute to the development and maturation of different types of GABAergic cells. Moreover, altered GABAergic signaling may contribute to phenotypes of autism, schizophrenia and epilepsy. Specific Cre-driver lines have begun to parcel out the functions of unique interneuron subgroups. Despite the advances in mouse models, it is often difficult to efficiently study GABAergic cortical interneuron progenitors with molecular approaches in vivo. One important technique used to study the cell autonomous programming of these cells is transplantation of MGE cells into host cortices. These transplanted cells migrate extensively, differentiate, and functionally integrate. In addition, MGE cells can be efficiently transduced with lentivirus immediately prior to transplantation, allowing for a multitude of molecular approaches. Here we detail a protocol to efficiently transduce MGE cells before transplantation for in vivo analysis, using available Cre-driver lines and Cre-dependent expression vectors. This approach is advantageous because it combines precise genetic manipulation with the ability of these cells to disperse after transplantation, permitting greater cell-type specific resolution in vivo.

Since MGE cells have the unique ability to migrate and integrate when transplanted into a host neocortex 16, they provide an excellent model system for genetic manipulation before in vivo studies. Herein, we show how one can isolate MGE tissue from E13.5 embryos (Figures 1 and 2), which can then be transduced with lentiviruses either in vitro or in a rapid manner before transplantation for in vivo studies. Labeling MGE via lentiviruses has been performed before using...

Watch this Scientific Journal Video about Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies at JoVE.com

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

GABAergic cortical interneuron progenitors disperse, develop and synaptically integrate into a host cortex after transplantation. These cells can be easily transduced before transplantation for in vivo studies of genetically modified GABAergic precursors. Here, we show viral labeling techniques to target specific interneuron subgroups using existing Cre lines and Cre-dependent reporters.

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically ...

viral-mediated-labeling-transplantation-medial-ganglionic-eminence

Research

JoVE Journal

Developmental Biology

11.4K Views.  University of California San Francisco. GABAergic cortical interneuron progenitors disperse, develop and synaptically integrate into a host cortex after transplantation. These cells can be easily transduced before transplantation for in vivo studies of genetically modified GABAergic precursors. Here, we show viral labeling techniques to target specific interneuron subgroups using existing Cre lines and Cre-dependent reporters.

Video: Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...