The overall goal of this technique is the assessment of the effect of gene expression manipulations on cortical neuronal architecture in vivo. Our first key advantage of this procedure over those based on a neutral electroporation, is the fine control of gene expression levels. Specifically, this integrated pipeline enables the in vivo evaluation of defect, exalted by small changes in gene expression on the control of neuronal cytoarchitecture.
Moreover, the pair structure of the transplantation test allows for a huge reduction in animal numbers necessary to obtain statistically significant results. The technique takes advantage of a precursor pool originating from Tau EGFP transgenic embryos. This pool is subdivided into two sub pools that are alternatively infected with two lentiviral mixes.
To obtain a green test pool and a yellow control pool that will be co-injected. After two days of in vitro expansion, the resulting neuro spheres are disassociated, mixed one to one, and injected with a capillary in the mouse ventricular space. Ten days later, immunofluorescence on coronal sections is performed to allow the comparison among test and control neurons.
Finally, the immunostained sections are imaged and skeletonized for final evaluation of morphometric parameters. Prior to in vivo transplantation, prepare the precursor green pool. E12.5 embryos, harvested from a pregnant dam previously mated to a Tau EGFP founder, are set in individual wells of a multi-well plate in PBS solution.
Genotype them quickly by visual inspection under a blue light lamp. Disassociate the dissected green cortices to single cells. Re-suspend cells in proliferative medium, and subdivide them into two equally-sized sub-pools.
Infect each sub-pool with a dedicated lentiviral mix. Each mix contains red label virus or black control virus. As well as viruses for Tet-Off driven transgene or control expression, respectively.
Each lentivirus must be administered at multiplicity of infection of 8. Plate the infected cells in proliferative medium with 2 micrograms per milliliter of doxycycline. Transfer cells to the incubator and leave them for 2 days at 37 degrees.
After 2 days, the two sub-pools must appear as suspensions of small neurospheres. Homogeneously expressing the red fluorescent protein, or not. After the engineering of precursors, set an assess array for the transplantation in P0 white lab pups.
Use the borosilicate glass capillaries with eternal and internal diameter equaling 1.5 millimeters and 1.12 millimeters, respectively. Pull the capillary with a P1000 puller. First, enter the pulling program.
Second, place the capillary into the holders and tighten them. Third, start the program and take the two pulled microcapillaries. Cut the capillary tip, by hand with a scalpel, under the stereomicroscope to obtain a tip with an external diameter of 200-250 micrometers.
Finally, place the capillaries on the plasticine support in a closed Petri dish, and transfer it under the hood. Disinfect optical fibers and place them under the hood. Mix the EGTA and Fast Green master mixes, to prepare the cell tracer solution, and place it again under the hood.
Cut small pieces of laboratory sealing film for cell suspension spotting. And finally, prepare a solution of 1 milligram per milliliter sterile doxycycline disposed in a 0.3 milliliter syringe. The injection tube is prepared from two latex tubes.
Fix a hard plastic mouth piece to one end of a latex tube. Then, fix the capillary holder to one end of the other latex tube. Connect the free ends of the two latex tubes from a 0.45 micrometer sterile filter, as a barrier against operator germs.
Place the resulting aspirator tube assembly on a plasticine holder to be kept under the hood. Collect, test, and control neurospheres in separate tubes. Centrifuge neurosphere suspensions and replace the supernatant by PBS.
Repeat this sequence two more times. Centrifuge neurosphere suspensions, replace the supernatant by trypsin solution, and pipette the cell pellet up and down, 4, 5 times. Leave cells in the incubator for five minutes to get a single cell suspension.
Block trypsin with trypsin inhibitor solution. Centrifuge cells, and re-suspend them in fresh proliferative medium. Count cells of the two pools and adjust their concentration to 100, 000 cells per microliter, in proliferative medium.
Then, mix test and control cells 1:1 and check the resulting mix under a fluorescence microscope. Finally, place the mix on ice under the hood. Prepare the cage with the mother and the pups on a table far away from the surgical operatory area.
Place a recovery cage on a cart. Put a mixture of sawdust taken from the mother's cage on it's bottom, and place the recovery cage under a lamp. Aside, set an ice box covered by an aluminum foil for the anesthesia of the P0 pups.
Just prior to the transplantation, add a 1 to 10 volume of cell tracer solution to one volume of cell suspension, and spot three microliters of the resulting mix on a piece of laboratory sealing film. Place the pup on the cool, aluminum foil for one minute, and check that it is fully anesthetized. Meanwhile, aspirate the 3 microliters of injection mix into the glass capillary.
Gently wipe the head of the anesthetized pup with 70%ethanol. Next, locate it on the optical fiber, to clearly identify the cortical hemispheres. Enter the capillary into the front and access the ventricular cavity.
While doing that, pay attention not to damage blood vessels. To prevent damage of ganglionic eminence, rotate the needle laterally by thirty degrees. Gently inject the cells into the cavity and monitor their diffusion by means of Fast Green.
Wait for five to ten seconds. Remove the glass needle, taking care not to aspirate the cell suspension, and discard it in a suitable disposal bin. Before pup recovery from anesthesia, inject 150 microliters of doxy solution intraperitoneally, to keep transgenic expression still off after the transplantation.
After the injection, place the pups immediately under the lamp for recovery. Leave the pups there for five to ten minutes, and once they are fully awake, place them in the cage with the mother. Observe the operated pups for two to three hours, to ensure that the mother accepts them.
Supply the cage with drinking water containing 0.5mg/mL doxycycline and sucrose, to maintain the transgenic expression off. Replace the doxycycline containing water with doxy-free water four days after transplantation. Thus activating the trans-gene in postmitotic neurons.
Keep mice in a doxy-free regime for six days, and euthanize them at P10. Fix brains from euthanized pups in 4%PFA, at four degrees, overnight. Remove the PFA solution and replace it with 30%sucrose solution.
Leave the brains at four degrees until they sink to the vial bottom. Transfer the brains into a disposable embedding mold approximately half filled with cryo-inclusion medium. Freeze the included brain at 80 degrees.
Cut 60 micron thick coronal sections using a cryostat. Process sections for anti-GFP, anti-RFP immunofluorescence and counterstain them with DAPI solution. Set working parameters of the confocal microscope appropriately as shown.
Collect pictures of immunoassayed slices, selecting GFP positive neuron-rich photographic fields blind of RFP signal distribution. Export the images as ND2 files and generate maximum z-projections of them as TIFF files. To allow for subsequent neuron skeletonization blind of cell genotype, hide the red signal.
To do so, add an adjustment layer. Select levels, select red. Set output to zero.
And save the file as such. Skeletonization is subsequently performed by a new operator, who had no previous access to original plain red files. For each hidden red file, open the file, add a drawing layer to the primary image, and select the pencil tool.
Trace the somer on the basis of GFP signal. Then, trace the neurites. Finally, save the mutlilayer file, including neuronal silhouettes.
Subsequent analysis of neuronal skeletons can be performed by either operator. Open each mulitlayer file, create new empty 16-bit grayscale files with black background. One per neuron.
Copy and paste single neuronal silhouettes, from the primary image, to the grayscale files. Get back to multilayer file and switch off the adjustment layer to unveil neuronal genotypes. Save the 16-bit grayscale files, annotating the corresponding neuron genotypes.
Import grayscale images in ImageJ one by one, and analyze skeletons by NeurphologyJ plugin. Collect selected NeurophologyJ primary data, total area of neurite length, attachment points and end points, into a spreadsheet, and employ them to compute secondary morphometric parameters. The engineering protocol we propose allows to cotransduce the vast majority of neurons subject of investigation, with the intended transgenes sets.
Moreover, it allows to achieve a substantial homogeneity of transgene expression levels within the transduced cell population. A key feature of our procedure, is it's paired configuration. A test and a control precursor pool are separately engineered, mixed 1:1, and finally co-transplanted.
This paired approach will facilitate the detection of results differences specifically linked to distinct genotypes. In this example, we analyzed coronal sections of transplanted pups, ten days after the interventricular co-injection of Foxg1 over-expressing neuron precursors and control ones. Images of groups of neurons were acquired with confocal microscopy.
Z-stack files were used to compute maximum projections. Neuronal silhouettes were manually traced. Here, green and yellow silhouettes represent Foxg1 over-expressing and control neurons, respectively.
Finally, primary parameters obtained by NeurphologyJ analysis, were employed for calculation of secondary morphometric indices, as illustrated. This is a simple protocol which allows the researcher to evaluate the impact that the mixed expression of a given gene, may exert on fine control of neurite development darth in vivo. The main advantages of this protocol are two.
Existing number of genes involved in neuronal morphogenesis may be functionally characterized in vivo, in the absence of the corresponding teratogenic lines. And fewer animals are needed for the analysis to get the significant statistical results.
