We thank Mihn Duc Do for his contribution to early setting up of this procedure.

All methods and procedures described here have been approved by the SISSA Organismo preposto al Benessere Animale (SISSA IACUC).
1. Generation of engineered &#34;green&#34; progenitor pools
<ol>
	<li>Preparation of the &#34;green&#34; pool
	<ol>
		<li>Mate a wild-type CD1 female with a MtaptEGFP/+&#160;founder16. Euthanize the pregnant dam by cervical dislocation at 12.5 days post-coitum (day 0 is determined by vaginal plug inspection) and harvest the embryonic day 12.5 (E12.5) embryos. Set them in individual wells of a 24 multiwell plate in cold PBS solution.</li>
		<li>Quickly genotype the embryos by visual inspection under a blue light lamp, checking for green fluorescence emission in the brains.</li>
		<li>Place the &#34;green&#34; embryos into a 10 cm diameter Petri dish filled with cold 1x PBS with 0.6% glucose and transfer it to under a stereomicroscope.</li>
		<li>Cut the embryo heads using standard scissors and extract the telencephalon from them by means of #3 and #5 forceps.</li>
		<li>Separate the two telencephalic vesicles and dissect out the neocortices using forceps; make sure to remove the hippocampus and the basal ganglia17.</li>
		<li>Collect neocortices by transferring them with a P1000 pipette into a 1.5 mL tube kept on ice.</li>
		<li>Let the dissected neocortices settle to the bottom of the tube.</li>
		<li>Aspirate the supernatant as much as possible.</li>
		<li>Resuspend the neocortices in 400 &#181;L of fresh proliferative medium [DMEM-F12, 1x Glutamax, 1X N2, 1 mg/mL bovine serum albumin (BSA), 0.6% glucose, 2 &#181;g/mL heparin, 20 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF), 1X pen-strep, and 10 pg/mL amphotericin B].</li>
		<li>Gently pipette the neocortices up and down, sequentially with P1000, P200, and P20 tips, 4x per tip, to obtain a cloudy cell suspension. 
		NOTE: E12.5 neocortical tissue is very soft; dissociating it to single cells does not require enzymatic digestion at all; repeated pipetting is sufficient.</li>
		<li>Let the meninges and residual neocortical clumps settle down for 2 min.</li>
		<li>Transfer 150 &#181;L from the top of the cell suspension (mainly containing single cells) into a new 1.5 mL sterile tube.</li>
		<li>Add 150 &#181;L of fresh proliferative medium and repeat steps 1.1.10-1.1.12 until no more tissue clumps are evident. While doing this, omit the P1000 pipetting step (in step 1.1.10). 
		NOTE: Three iteration of steps 1.1.10-1.1.12 are usually sufficient to achieve full tissue dissociation.</li>
		<li>Count cells (&#34;green&#34; pool) with the trypan blue exclusion method in a B&#252;rker chamber17 and add fresh proliferative medium to adjust the concentration to 103 cells/&#181;L. 
		NOTE: Steps 1.1.7-1.1.14 must be performed under a sterile laminar flow hood disinfected by 70% ethanol and kept ventilated for at least 20 min.</li>
	</ol>
	</li>
	<li>Engineering the &#34;green&#34; sub-pools
	<ol>
		<li>Subdivide the &#34;green&#34; pool into two sub-pools in two distinct 1.5 mL tubes for lentiviral infection.</li>
		<li>Infect the sub-pools independently with the two dedicated lentiviral mixes. Each mix contains &#34;red label&#34; virus (Pgk1_mCherry) or &#34;black&#34; virus (pCAG_lacZ) as a control, as well as viruses for tetOFF driven transgene (Pgk1p_tTA and TRE_transgene) or control (Pgk1p_tTA and TRE_PLAP) expression. 
		CAUTION: Manipulate lentiviruses in a BLS-2 environment with all the protective measurements prescribed by applicable rules and laws. 
		NOTE: Each lentivirus must be administered at a multiplicity of infection (MOI) of 8, which (as previously shown14) is sufficient to transduce the vast majority of neural cells in such experimental conditions (Figure 1). The lentivirus harboring tet transactivators can be built by employing different neuronal promoters driving tTA/rtTA expression (e.g., pT&#945;1, pSyn, pCaMKII, pGAD1, etc.) (Figure 2). The MOI is the ratio of (a) the number of infectious viral particles delivered to (b) the number of their target cells. Here, the former (a) is calculated according to the conventional procedure described elsewhere14, the latter (b) is simply evaluated using a B&#252;rker chamber.</li>
		<li>Plate 600,000 cells of each infected sub-pool per well of a 12 multiwell plate, in 600 &#181;L of proliferative medium with 2 &#181;g/mL of doxycycline. 
		NOTE: Here, wells are not pretreated with poly-lysine, which prevents neural cell attachment to their bottoms.</li>
		<li>Transfer cells to the incubator in 5% CO2 at 37 &#176;C.</li>
		<li>After 2 days, to assess the efficiency of infection and the differential marking of the engineered pools, inspect the cells under a conventional fluorescent microscope. The two sub-pools must appear as suspensions of small neurospheres, homogenously expressing the red fluorescent protein (&#34;control&#34; sample) or not (&#34;test&#34; sample). Within these neurospheres, the MtaptEGFP transgene is active limited to a minority of cells (post-mitotic neurons) and silent in the majority of them (proliferating precursors) (Figure 3).</li>
	</ol>
	</li>
</ol>
2. Setting up of the surgical instruments and the operating area
<ol>
	<li>Preparing borosilicate needles
	<ol>
		<li>Use the borosilicate glass capillaries with external and internal diameter equaling 1.5 mm and 1.12 mm, respectively.</li>
		<li>Pull the capillary with a P 1000 puller.</li>
		<li>Set up the pulling program. Typical program parameters are: heat = 614; vel = 60; time = 1; pressure = 600. They must be adjusted according to the puller model and the heating resistor employed.</li>
		<li>Place the capillary into the puller holders and tighten them.</li>
		<li>Start the program and take the two pulled microcapillaries.</li>
		<li>Cut the capillary tip by hand, with a scalpel, under the stereomicroscope to obtain a tip with an external diameter of ~200-250 &#181;m.</li>
		<li>Place the capillaries on a modelling clay (e.g., plasticine) support in a closed Petri dish and transfer it to under the hood.</li>
	</ol>
	</li>
	<li>Setting up the hood and preparing the cell tracer solution
	<ol>
		<li>Clean up the horizontal flow hood carefully and disinfect it using 70% ethanol.</li>
		<li>Disinfect optical fibers using 70% ethanol and place them under the hood.</li>
		<li>Mix 10 &#181;L of 125 mM EGTA solution and 10 &#181;L of Fast Green to prepare the &#34;cell tracer solution&#34; and place it under the hood.</li>
		<li>Cut small pieces (4 cm x 2 cm of size) of laboratory sealing film for cell suspension spotting.</li>
		<li>Prepare a solution of 1 mg/mL sterile doxycycline and aspirate it into a 0.3 mL syringe.</li>
		<li>Switch on the hood and keep the laminar flow on for 20 min before the operation.</li>
	</ol>
	</li>
	<li>Assembling the injection tube
	<ol>
		<li>Take a hard-plastic mouthpiece, two latex tubes (each about 30 cm long) and a capillary holder from an aspirator tube assembly kit for calibrated microcapillary pipettes.</li>
		<li>Fix the hard-plastic mouthpiece to one end of a latex tube.</li>
		<li>Fix the capillary holder to one end of another latex tube.</li>
		<li>Connect the free ends of the two latex tubes with a 0.45 &#181;m sterile filter, as a barrier against operator germs.</li>
		<li>Place the resulting aspirator tube assembly on a modelling clay holder to be kept under the hood.</li>
	</ol>
	</li>
</ol>
3. Cell mix and intraventricular transplantation
<ol>
	<li>Mixing &#34;test&#34; and &#34;control&#34; green cell sub-pools
	<ol>
		<li>Collect &#34;test&#34; and &#34;control&#34; neurospheres (see step 1.2.5) in separate 1.5 mL tubes.</li>
		<li>Centrifuge neurospheres suspension at 200 g for 5 min.</li>
		<li>Collect and discard the supernatant, avoiding disturbance of the cell pellet.</li>
		<li>Gently resuspend neurospheres in 500 &#181;L of 1x PBS.</li>
		<li>Centrifuge them at 200 g for 1 min.</li>
		<li>Remove the supernatant.</li>
		<li>Repeat steps 3.1.4-3.1.6 two more times.</li>
		<li>Gently resuspend spheres in 200 &#181;L of 2x Trypsin solution (2x Trypsin, 1x PBS) and pipette cell pellet up and down 4x-5x. 
		NOTE: Pay attention not to make air bubbles.</li>
		<li>Leave cells in the incubator for 5 min at 37 &#176;C to achieve a single-cell suspension.</li>
		<li>Block Trypsin with 200 &#181;L of Trypsin inhibitor solution (DMEM-F12, 10 &#181;g/mL DNAse I, 0.28 mg/mL Trypsin inhibitor) by pipetting up and down 4x-5x.</li>
		<li>Centrifuge cells at 200 x g for 5 min and resuspend them in 1 mL of fresh proliferative medium (DMEM-F12, 1x Glutamax, 1x N2, 1 mg/mL BSA, 0.6% glucose, 2 &#181;g/mL heparin, 20 ng/mL bFGF, 20 ng/mL EGF, 1x pen-strep, 10 pg/mL amphotericin B).</li>
		<li>Count cells of the two pools with trypan blue exclusion method in a B&#252;rker chamber.</li>
		<li>Adjust their concentration to 100,000 cells/&#181;L in proliferative medium.</li>
		<li>Mix 1:1 &#34;test&#34; cells with &#34;control&#34; ones.</li>
		<li>Check the resulting mix under a fluorescence microscope (objective magnification: 10x) to ensure that the mix is a single-cell suspension of the two pools (Figure 3C).</li>
		<li>Place the mix on ice under the hood.</li>
	</ol>
	</li>
	<li>Intraventricular transplantation
	<ol>
		<li>Prepare the cage with the mother and the P0 pups on a table far away from the surgical operatory area.</li>
		<li>Place a recovery cage on a cart close to the hood.</li>
		<li>Put a mixture of sawdust taken from the mother&#39;s cage on the bottom of the recovery cage and place it under a lamp.</li>
		<li>Aside, set an ice box covered by an aluminum foil for the anesthesia of P0 pups.</li>
		<li>Just prior to transplantation, add 1/10 volume of cell tracer solution (from step 2.2.3) to 1 volume of cell suspension (from step 3.1.16) and spot 3 &#181;L of the resulting &#34;injection mix&#34; on a piece of laboratory sealing film.</li>
		<li>Place the pup on the cool aluminum foil for 1 min and check that it is fully anesthetized. 
		NOTE: To confirm anesthesia, gently squeeze a paw and monitor the pup for lack of movements, while still breathing.</li>
		<li>Meanwhile, aspirate the 3 &#181;L of injection mix (from step 3.2.5) into the glass capillary.</li>
		<li>Gently wipe the head of the anesthetized pup with 70% ethanol.</li>
		<li>Place the pup&#39;s chin on the tip of the optical fiber to clearly identify the cortical hemispheres. 
		NOTE: At P0-P1, the head skin is very thin, allowing for straightforward visual identification of the hemispheres just above the eyeballs and behind the olfactory bulbs.</li>
		<li>Keep the capillary (loaded as described in step 3.2.7) in either one of the two mid-parasagittal planes (left or right), above the olfactory bulb, and puncture the forehead skin at its intersection with the frontal plane containing the eyeballs centers. Pay attention not to damage blood vessels. Enter the capillary into the frontal cortex and access the ventricular cavity (Figure 4A).</li>
		<li>To prevent accidental damage of the ganglionic eminence, rotate the needle laterally by 30 degrees, pointing its tip caudal-medial-ward (Figure 4B).</li>
		<li>Gently inject the cells into the ventricular cavity and monitor their diffusion by means of Fast Green (Figure 4C).</li>
		<li>Wait 5-10 s.</li>
		<li>Remove the glass needle taking care not to aspirate the cell suspension and discard it in a suitable disposal bin.</li>
		<li>Before pup recovery from anesthesia inject 150 &#181;L of doxycycline solution intraperitoneally to keep transgene expression still off after the transplantation.</li>
		<li>After the injection, place the pup immediately under the lamp for recovery.</li>
		<li>Leave the pups under the lamp for 5-10 min and, once fully awake, place them in the cage with the mother.</li>
		<li>Observe the operated pups for 2-3 h, to ensure that the mother accepts them.</li>
		<li>Supply the cage with drinking water containing 0.5 mg/mL doxycycline and 50 g/L sucrose to maintain transgene expression off.</li>
		<li>Replace the doxycycline-containing water by doxy-free water 4 days after transplantation, thus activating the transgene in post-mitotic neurons. Keep mice in a doxy-free regime for 6 days and euthanize them at P10 by carbon dioxide inhalation followed by decapitation.</li>
	</ol>
	</li>
</ol>
4. Analysis of transplanted brains
<ol>
	<li>Histology and immunofluorescence
	<ol>
		<li>Euthanize the transplanted pups 10 days after cell transplantation by carbon dioxide inhalation followed by decapitation. Fix brains of euthanized pups in 4% paraformaldehyde (PFA) at 4 &#176;C overnight. 
		CAUTION: PFA is highly toxic; handle it with care, strictly complying with manufacturer&#39;s prescriptions, under a chemical fume hood; discard PFA solution residual in a labeled waste container.</li>
		<li>Remove the PFA solution and replace it with 30% sucrose solution.</li>
		<li>Leave the brains at 4 &#176;C or until they sink to the vial bottom.</li>
		<li>Transfer the brains into a disposable embedding mold approximately half-filled with cryo-inclusion medium.</li>
		<li>Freeze the included brains at -80 &#176;C.</li>
		<li>Cut 60 &#181;m thick coronal sections using a cryostat. 
		NOTE: Avoid employing thinner sections, as this may result in unacceptable loss of information regarding the whole architecture of single neurons.</li>
		<li>Process sections for immunofluorescence18,19, using anti-GFP [1:400] and anti-RFP (mCherry) [1:500] antibodies. Counterstain nuclei with 1 mg/mL DAPI solution [1:200].</li>
	</ol>
	</li>
	<li>Morphometry
	<ol>
		<li>Set working parameters of the confocal microscope as follows: z-stack height = 40 &#181;m; step = 2 &#181;m.</li>
		<li>Collect pictures of immunoassayed slices selecting GFP positive neuron-rich photographic fields, blind of RFP signal distribution.</li>
		<li>Export the images as .nd2 files.</li>
		<li>Generate maximum Z-projections of them as .tiff files (Figure 5A).</li>
		<li>To allow for sub-sequential neuronal skeletonization blind to cells genotype, hide the red signal. To do so, open each .tiff file by a suitable software and add an adjustment layer. Select &#34;levels&#34;, &#34;red&#34;, then set &#34;output&#34; to zero. Save the file as such. 
		NOTE: Skeletonization is subsequently performed by a new operator who had no previous access to original plain red files.</li>
		<li>For each hidden red file, add a drawing layer to the primary image and select the pencil tool (white color). Trace the soma on the basis of GFP signal, then trace the neurites. 
		NOTE: the pencil tool may be set at 40 pixels for the soma and at three pixels for neurites.</li>
		<li>Save the multilayer file including neuronal silhouettes (Figure 5B). 
		NOTE: Subsequent analysis of neuronal skeleton can be performed by either operator.</li>
		<li>Create a new 1024 x 1024 16-bit grayscale file with black background, one per neuron.</li>
		<li>Copy and paste single neuronal silhouettes from the primary image to the new grayscale file. Get back to multilayer file and switch off the adjustment layer to unveil neuronal genotypes. Save the 16-bit grayscale file annotating the corresponding neuronal genotype.</li>
		<li>Import grayscale images in ImageJ one by one and analyze skeletons by NeurphologyJ plugin of ImageJ software20 (Figure 5C).</li>
		<li>Copy NeurphologyJ primary data (neurite_length, attachment_points and endpoints; for each, take &#34;Total Area&#34; values), paste them into a spreadsheet and employ them to compute secondary morphometric parameters (average neurite length, branching index) (Figure 5D).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Specific aspects/steps of this procedure are critical and require special attention. First, (a) operators must be adequately pretrained to safely manipulate lentiviruses in a BSL-2 compliant lab environment. Second, (b) prior to mix &#34;test&#34; and &#34;control&#34; neural preparations, it is mandatory to carefully wash the two corresponding neurosphere suspensions as described, in order to prevent any delayed cross-infection of the two preparations due to unwanted lentiviral carry over. Third, (c) while transplanting...

Here we describe a simple method we developed to dissect in vivo gene control of neuronal cytoarchitecture. Based on in vitro engineering of neuronal precursors, their transplantation into neonatal brain and paired morphometric evaluation of &#34;test&#34; and &#34;control&#34; cells, it allows to unveil functional implications of test genes in fine control of neuronal morphology in a fast and affordable way. To investigate in vivo&#160;gene control of neuronal architecture, three key technical issues must be addressed: (1) achieving an adequately patterned gene-of-interest (GOI) expression and an accurate quantitative control of it; (2) obtaining a properly segmented visualization of distinct neuronal silhouettes; (3) eliciting statistical significance of results while employing a limited number of animals.
When available, mouse mutant lines harboring tetracycline (tet)-controlled transgenes may be the best tool to address the first issue1. Alternatively, somatic transgenesis may be employed. In such cases, the transgene is delivered via electroporation2 or viral transduction3. Next, it is retained as an episome (e.g., upon standard electroporation4), or it is integrated into the genome (randomly, via retroviral integrase5; or in a defined location, via CRISPR-promoted homologous recombination (SLENDR)6).
Second, neuronal silhouette visualization may be achieved via (a) sparse uniform labeling or (b) dense differential labeling. As for sparse labeling, advanced Golgi-like methodologies may be employed7, selected neuronal minisets can be filled by biocytin8, and salt-and-pepper labeling can be obtained thanks to a sparsely expressed transgene. Such a transgene may display variegated transcription (Thy-EGFP)9 or may be activated by stochastic recombination (MORF)10. As for (b), state of art strategies include Cre-mediated stochastic recombination within a multi-floxed fluoroprotein transgene array (Brainbow)11, as well as piggyBac-transposase-driven genomic integration of fluoroprotein genes, previously delivered via somatic transgenesis (CLoNE)12.
As for the third issue, the morphometric outcome is often affected by a large random variability, originating from inter-animal differences and cell-injection contingencies. Because of this, a large number of animals is usually employed to achieve the statistical power necessary to assess GOI morphometric activity.
The approaches previously described often rely on advanced technical skills and require conspicuous financial resources, which may limit their diffusion within the scientific community. To circumvent these issues, we conceived an easy and straightforward pipeline to dissect gene control of neuroarchitecture in vivo in a fast and affordable way. This is inspired by a similar co-transplantation design previously developed for fast in vivo evaluation of antiblastic transgene activity13.
Specifically, it is thought that the co-transplantation of in vitro engineered &#34;green&#34; neural precursors (&#34;test&#34; and &#34;control&#34; cells) into a &#34;black&#34; recipient neonatal brain can simultaneously fix the three key issues listed above. In fact, in vitro lentiviral engineering of precursors, in well-controlled conditions, allows maintenance of variability of neuronal transgene expression at a minimum, far below that usually associated with in vivo somatic manipulations (performed previously14,15 and in our unpublished results). The resulting accurate control of gene expression is comparable with that achieved by tet-controlled transgenic models. However, costs of this procedure are far below those originating from maintenance of a transgenic mouse line. Next, free-hand cell injection is easy and requires minimal training. Moreover, the amount of labeled precursors injected into each brain can be easily tuned to achieve a sufficient cumulative number of sparsely distributed precursors, while also keeping the total number of transplanted animals at a minimum. Last but not least, co-injection of differently fluoro-labeled, &#34;test&#34; and &#34;control&#34; precursors and subsequent pairwise statistical analysis of the results counteract the effects of inter-animal experimental variability, allowing the reaching of statistical significance of results, even upon the analysis of a limited number of individuals13.
It should be emphasized that, albeit fast and cheap, this method has two main limitations. First, it is designed to investigate cell-autonomous gene control of neuronal architecture, and it is not appropriate to address environmental control. Second, as transplanted neuronal precursors reach their final location by a heterochronic schedule, this method is preferable to model neuroarchitectonics control occurring past migration completion.

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intraventricular transplantation of engineered neuronal precursors for in vivo neuroarchitecture studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

There are five primary datasets providing useful information about key aspects of the procedure, the first being (1) efficiency of neural precursors transduction and co-transduction by lentiviral vectors. (2) An example of key features of the promoters employed to drive the &#34;test gene&#34;. (3) An example of engineered cells ready for transplantation. (4) A cartoon including key procedural details of cell microinjection into the neonatal brain. (5) A synopsis of the whole morphometric...

Watch this Scientific Journal Video about Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies at JoVE.com

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

intraventricular-transplantation-engineered-neuronal-precursors-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

5.6K Views.  SISSA. Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Video: Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

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Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila