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

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

After preparing lentivirus and dissecting the MGE according to the directions in the written protocol, collect both hemispheres of the MGE and put the tissue into two 1.5-milliliter collection tubes containing 500 microliters of DMEM with 10% FBS.
Keep the samples on ice until all tissue is collected. Then transfer the tubes to a BSL-2-certified hood. Carefully aspirate the media without disturbing the MGE tissues settled at the bottom of the tube.
Then add around 500 microliters of pre-warmed DMEM with 10% FBS media. Add polybrene to a final concentration of 8 micrograms per milliliter, and titrate through a P1000 pipette tip until a single-cell suspension is achieved. Then, add 15 to 20 microliters of the concentrated lentivirus to each tube.
Next, securely close each tube, invert to mix, and then place all tubes in a 37 degrees Celsius incubator. Incubate the cells with the lentivirus for at least 30 minutes, but not longer than an hour, as longer times have resulted in decreased cell viability. Invert the tubes every 10 minutes.
Following the incubation, remove the tubes from the incubator, and centrifuge at 700 times g at 4 degrees Celsius for three minutes to pellet the cells. In the BSL-2 hood, remove the supernatant and discard into 10% bleach. Next, add 1 milliliter of DMEM with 10% FBS, and titrate the pellet two to three times to wash.
After centrifuging as before, repeat this wash step two to three more times to remove excess virus. 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, approximately 2 to 3 microliters of media will end up covering the cell pellet, by the time the transplantation procedure has begun. While ensuring that sterile technique is used throughout, draw up mineral oil into a 1-milliliter syringe, and then with a 30.5 gauge needle, back-fill the glass pipette completely with the mineral oil.
Next, attach the glass pipette to the stereotaxic device and load onto the plunger. With an assistant using the hydraulic drive, move the plunger about halfway into the glass pipette, removing the mineral oil that spills out with a clean paper towel.
Next, twist the corner of a sterile paper towel into a fine point, and use this to delicately remove excess media above the MGE cell pellet. This will concentrate the cell density so that smaller injection volumes can be achieved.
Next, use a P2 pipette to slowly draw up the cell pellet and titrate one to two times before dispensing onto a hydrophobic surface. The volume should be just under 1 microliter.
Move the tip of the needle into the cell suspension, and using the hydraulic drive, draw up the cell suspension into the pipette. After anesthetizing a pup via hypothermia, check for the effectiveness of anesthesia by pinching the skin between the toes with forceps. No reaction should be seen.
Next, place the pup onto a mold that is under the injection device, and make the skin taut by pulling back the skin on the head and then securing with standard lab tape. Working quickly, manipulate the stereotaxic arm to position the tip of the glass pipette on the pup&#39;s head, perpendicular to the surface of the head.
When the pipette is in contact with the head, consider this a zero. Next, push the pipette through the surface of the skin and skull into the cortex. Then, insert and retract the pipette two times before stopping.
For targeting of cells into the neocortex, injections are performed when the micropipette is at a depth of 0.1 millimeter. Once the needle is in position, rotate the hydraulic device to advance the plunger into the glass pipette, and inject 50 to 70 nanoliters per site.
Repeat this procedure at three to six different sites at different rostral-caudal levels if a wide distribution of transformed cells throughout the neocortex is required. When injections are complete, remove the pup from the stage and mark it according to approved procedures.
Once the pup has recovered and is able to move on its own, put it back with the lactating female and the rest of the litter. Check the pups after the procedure and the following day, to ensure that there are no signs of deteriorating health.

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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents and equipment for MGE cell transplantation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>REF 309602</td>
			<td>BD, Becton Dickinson and company</td>
			<td></td>
		</tr>
		<tr>
			<td>301/2&#160;gauge needle</td>
			<td>305106</td>
			<td>BD, Becton Dickinson and company</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision bore to deliver 5 &#181;l (comes with plunger)</td>
			<td>5-000-1005</td>
			<td>Drummond scientific company</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>PM-999</td>
			<td>Polysciences, Inc.</td>
			<td></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>Stoelting company</td>
			<td></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>made in house</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle pipette puller</td>
			<td>730</td>
			<td>Kopf instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>n.a.</td>
			<td>Any drug store or pharmacy</td>
			<td></td>
		</tr>
		<tr>
			<td>Any pipette and tips that can reliaby measure 1 &#181;l of volume</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>** These are the particular models we use, but many other setups should work</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optional Reagents (for making Lentiviruses)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 mm, 0.45 &#181;m filter</td>
			<td>09-719B</td>
			<td>Fisher Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear centrifuge tubes (25 x 89 mm)</td>
			<td>344058</td>
			<td>Beckman Coulter&#174;</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine&#160;2000&#160;(1.5 ml size)</td>
			<td>11668019</td>
			<td>Invitrogen</td>
			<td></td>
		</tr>
		<tr>
			<td>Other commercially available supplies</td>
			<td></td>
			<td></td>
			<td></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>

Source: Vogt, D. et al., <a href="https://app.jove.com/v/52740">Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies.</a>&#160;J. Vis. Exp.&#160;(2015).
This video demonstrates the viral labeling and transplantation of medial ganglionic eminence (MGE) cells for in vivo studies. It outlines the steps involved in transducing MGE cells with a viral reporter, transplanting these transduced cells into the cortex of a mouse pup, and enabling reporter expression in specific interneuron subtypes.

<img alt="Figure 1" src="/files/ftp_upload/23124/23124_Figure1.jpg" /> 
Figure 1.&#160;Representative procedure for the dissection of E13.5 MGE tissue.&#160;Example dissection of MGE tissue for either primary cultures or transplantations.&#160;(A-E)&#160;Images of E13.5 brain dissections, and&#160;(A&#39;-E&#39;)&#160;schematics to highlight structures and important steps in the procedure.&#160;(A, A&#39;)&#160;Repre...

Source: Vogt, D. et al., Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies.&#160;J. Vis. ...

Harvest the medial ganglionic eminence tissue, containing cortical interneuron progenitors, from genetically modified mouse embryos.&#160;
Add a cationic polymer and dissociate the tissue into a single-cell suspension.
Add lentiviral vectors carrying a Cre recombinase-dependent GFP reporter flanked by lox sites. Incubate.
The polymer neutralizes the viral- and host-cell-membrane charges, enabling viral fusion and RNA release.
The viral RNA is reverse-transcribed and integrated into the host genome.
Centrifuge and discard the supernatant. Absorb excess media.
Transfer the cells to a hydrophobic surface and load them into an injection device.
Inject the cells into an anesthetized mouse pup&#39;s cortex.
Allow the pup to recover and grow.
The transplanted progenitors differentiate into mature interneurons in the cortex.
A subgroup of these interneurons co-expresses Cre recombinase and RFP.
In these interneurons, Cre excises the lox sites, flipping the GFP gene and enabling its expression.
Visualize the RFP and GFP co-expression to identify Cre-expressing interneurons.

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视频: Viral-Mediated Labeling and Transplantation of Medial Ganglionic Eminence Cells for In Vivo Studies - 实验

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.

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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 ...

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发育生物学

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

离体 小鼠尿路上皮细胞腺病毒-Cre介导的基因缺失的类器官模型

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

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Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

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

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