We thank Birgit Westernstroeer of the Centre of Reproductive Medicine and Andrology at the University of Muenster for teaching the testicular microinjection. Besides, we are grateful to Ursula Antkewitz and Petra Reckling for technical assistance. We thank the German Research Foundation (DFG) for supporting this work (WE2458/10-1).&#160;

All performed animal experiments have been approved by the local ethics committee (Landesamt f&#252;r Landwirtschaft, Lebensmittelsicherheit und Fischerei, Mecklenburg-Vorpommern, Germany).
1. Plasmid Preparation
<ol>
	<li>For plasmid preparation, use plasmid purification kits (see Materials table) or similar methods with endotoxin removal buffer so that immune reactions of the animal can be avoided. Follow the instructions of the manual. Employ ddH2O to dilute the plasmid solution.</li>
	<li>Eliminate debris by spinning the DNA solution at maximum speed (20,000 x g). Then collect the supernatant.</li>
	<li>Determine plasmid concentration with a spectrophotometer (see materials).</li>
	<li>Adjust plasmid concentration with ddH2O to 1-3 &#181;g(DNA)/&#181;l.&#160;Note: Lower concentrations will reduce transfection efficiency, while higher concentrations might cause a too viscous injection solution. Besides, the transfection efficiency depends on the size of the plasmid and has to be tested individually.</li>
	<li>Prior to injection, prepare a solution mix with for example&#160;40 &#181;l plasmid together with 5 &#181;l PBS (10x) and 5 &#181;l Fast Green (0.5%). Fast Green is needed for tracking the injection process. Preferably, use thin wall PCR-tubes or Parafilm. Note:&#160;Depending of the size of the testis, a volume of 20-50 &#181;l will be needed for each testis.&#160;</li>
</ol>
2. Preparation of Microinjection Pipette
<ol>
	<li>Use borosilicate capillaries (see &#160;Materials table) for preparing the microinjection pipettes.</li>
	<li>Pull glass capillaries with a vertical capillary puller (see&#160;Materials table).</li>
	<li>Break the capillary tip with forceps by softly banding. The tip is usually 50-80 &#181;m in diameter and about 1 cm long. Try to avoid tips that are too long as these are not stiff enough to penetrate the tissue.</li>
	<li>At last, sharpen the tip in a 30&#176; to 45&#176; angle by using a micropipette beveler (see&#160;Materials table).</li>
	<li>Load the microinjection pipette with the injection solution mix (see Plasmid Preparation). Apply the solution into the back of the glass capillary with a small syringe. Note: Try to avoid air bubbles while loading, otherwise these will be injected into the seminiferous tubules along with the plasmid solution and hence will reduce conductivity as well as possibly cause tissue damage.</li>
</ol>
3. Anesthesia and Surgery
<ol>
	<li>Perform the operation under aseptic conditions by using sterile material (i.e., syringe, needle, surgical instruments, etc.). This will reduce infection of the animal and ensure good survival rates.</li>
	<li>Use male mice for electroporation at an age of 6-8 weeks.</li>
	<li>To initialize postsurgical analgesic treatment, provide the mouse with analgesics added drinking water on the day prior to surgery. This assures a preemptive analgesia in case of highly probable postsurgical hypodipsia (diminished water intake). To this end, expose drinking water with for example a pediatric ibuprofen suspension (20 mg/ml). The medicated drinking water should also be supplied on day one and two of recovery in identical concentration. This means a daily dose of 7.5 mg/kg, valid for 5 ml/d drinking water and body weight of 25g for 8 weeks aged C57BL/6J. This analgesic regimen guarantees a fundamental pain relief and accelerated recovery along with high welfare26.</li>
	<li>For preparing&#160;the anesthesia working solution on the day of surgery, mix 10% Ketamin and 2% Xylazin in a 1:1 ratio (see&#160;Materials table).</li>
	<li>To anesthetize the animal, apply 0.25 ml/100 mg body weight of anesthetic solution subcutaneously (Ketamin = 0.125 g/kg; Xylazin = 0.025 g/kg). Use an injection site between pelvic and limb in order to prevent organ damage and injury of the mice. Usually, 10 min are needed until the mouse is deeply anesthetized.</li>
	<li>Cover the mouse eyes with vet ointment to prevent dryness during anesthesia.</li>
	<li>Test deep anesthetic arrest, which is noticeable by a total lack of response. To this end, just pinch the toe of the animal.</li>
	<li>To start the surgery, remove abdominal hair with an electric shaver or similar and disinfect the operation area with sterilium or similar.</li>
	<li>Make a ventral incision directly above the preputial glands in the center of the abdominal area. At that place, first pull the skin away and conduct a small transversal cutaneous cut of about 8-14 mm. Then, continue along the abdominal muscular layer. 
	Note: The lesion should be as small as possible to reduce harm to the animal.</li>
	<li>Pull up the abdominal fat pads carefully to expose the attached testis and place it on a prior prepared waterproof disposable paper drape just beside the incision site&#160;(Figure 2A).</li>
	<li>Use binoculars to find the efferent duct as the injection target. The ductus efferent is identified as the fine vessel junction between the testis and the epididymis. It is located adjacent to the prominent testicular artery. The ductus runs almost at an angle of 45&#176; to the artery and visibly enters the caput epididymidis. Note: Depending on the mouse&#8217;s age, the ductus is usually buried in fatty tissue.</li>
	<li>Use fine forceps to clear the efferent duct of this fatty tissue. After that, place the released ductus on a sterile&#160;paper strip to ensure the clear visibility of the duct without impairing surroundings.</li>
</ol>
4. Microinjection and DNA Application
<ol>
	<li>Connect the microinjection pipette, which is loaded with the plasmid solution to the micromanipulator/injector unit.</li>
	<li>Place the microinjection needle parallel to the efferent duct with the tip pointing towards the rete testis (Figure 2B).</li>
	<li>Use fine tweezers and strip the duct over the microinjection pipette. Make sure that the capillary is kept parallel to the duct while pulling it over. 
	Note: This procedure is more convenient than penetrating the vessel by moving the needle with the micromanipulator.</li>
	<li>Direct the needle carefully towards the testis and stop just as it penetrates the rete testis directly beneath the tunica albuginea (Figure 2C). 
	Note:&#160;In the case of overreaching the rete testis, the plasmid solution will leak into the interstitial space. This commonly leads to failure of transfecting the seminiferous tubules.</li>
	<li>For injection of the plasmid solution utilize the microinjector with following settings: pi:&#160;100 hPa, ti:&#160;0.2 sec, and pc:&#160;0 hPa (Figure 2D).</li>
	<li>Monitor the entire injection process by observing the testis filling status with the help of the green color. Take care that the testis is only filled up to 2/3 of its volume with the plasmid solution (Figure 2E). 
	Note:&#160;If the injection volume is exceeded, the testis tissue could be harmed.</li>
</ol>
5. Electroporation of Testis
<ol>
	<li>To enable effective electroporation, soak the tweezer electrodes in PBS (1x). This ensures adequate conductance.</li>
	<li>Smoothly squeeze the testis between the wet electrodes (Figure&#160;2F). Measure the electrical resistance with the electroporator.</li>
	<li>Apply current to the testis. Perform eight square pulses of 40 V at 4&#160;different testis sites with a constant duration of 50 msec pulse time and 950 msec interval time.</li>
</ol>
6. Wound Closure and Post-surgery
<ol>
	<li>When electroporation is finished, place the testis back in the original location.</li>
	<li>Sew the inner muscular layer with surgical suture (see materials).</li>
	<li>Close the skin by employing suture clips (see Materials table). Pull the skin up with tweezers. Make sure to exclude the muscular layer. Place the clips, each at a distance of not more than 5 mm. 
	Note: Because surgical suture can be swallowed by the mouse, suture clips are favored for closing the skin lesion.</li>
	<li>Allow the mouse to recover from anesthesia on sterile paper towels placed in a sterile empty mouse cage, which is tempered on a 37 &#176;C warm pad. The pad should ensure the warming of one half of the cage creating a heat gradient. For the animal this makes possible to search for the individual, most comfortable area due to the temperature variety in the cage.&#160;&#160;In addition, cover the mouse with a sheet of sterile paper towel so that stress can be further reduced. Note:&#160;Since the mouse&#8217;s body surface is uncommonly high in relation to body mass, when compared with larger animals, the thermal support is of particular importance for successful recovery of rodents.</li>
	<li>When the operated animal has fully awakened, transfer it for further recovery to a completely equipped new mouse cage. Do not put the animal back to its old cage or to a group of mice. Ensure that the cage is as clean and sterile as possible to prevent wound infection. Monitor the entire recovery process. To transfer the mouse back to the animal care facility, wait until it has fully recovered and appears to behave normally. 
	Note: Additional per- and post-surgical strategies are discussed by Pritchett-Corning KR et al6.</li>
</ol>

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Marten Michaelis, Alexander Sobczak, and Joachim M. Weitzel employed at the Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany, declare that they have no competing financial interests.

Research in the field of reproductive biology, particularly in the area of male fertility and spermatogenesis inevitably relies on living organisms. In order to examine testicular function, no adequate cell culture/in vitro system has been established capable of reflecting all the crucial morphological changes from a diploid spermatogonium to a haploid mature spermatozoon1,2. Thus, the generation of genetically modified animals is often a necessary and as such a valuable tool in male ...

Mammalian spermatogenesis is considered to be a sophisticated process of self-renewing stem cells successively undergoing mitosis, meiosis and differentiation in order to develop into mature haploid spermatozoa. These morphological changes are orchestrated by different cell types and despite profound attempts, it is still impossible to mimic these processes in cell culture1,2. Hence, research on spermatogenesis up to now relies on living organisms as in vivo models. In general, gene function studies are usually based on transgenic animals. However, generating and sustaining this kind of animal model is time-consuming, cost-intensive and quite elaborate. This is attributed to the required long breeding process for generating and maintaining the transgene over the generations. Additionally, the genetic manipulation of the entire organism by the transgenic or knockout approach is prone to cause physiological impairments when targeting genes with essential functions in multiple regions, e.g., outside the testis or systemically.
Further, some transient transfection methods are associated with some crucial disadvantages. For example, typical drawbacks of virus-mediated gene transfer are the possible provocation of immunoreactions and additional safety regulations, whereas lipofection3 and microparticle bombardment4 might damage the tissue and are limited to a certain cell depth for sufficient transfection efficiency.
In contrast, electroporation (EP) as another common way of transient transfection, seems to constitute a promising technique for enabling in vivo transfection and consistent in vivo gene analysis. In general, EP is referred to as a dynamic phenomenon that depends on local transmembrane voltage with consequently mediated pores within nanoseconds. These gaps can be maintained for milliseconds, sufficient to grant access to DNA, RNA or small molecules5. When the applied voltage&#160;is too high, the usually transient character of EP is counteracted due to heat production and induction of too comprehensive permeabilization with consequent irreversible damage of the cell5.
Here, we show that electroporation is an effective and economical transfection system which is capable of being utilized for genetic testis transformation in order to elucidate testicular gene characteristics in vivo. This article addresses plasmid preparation, microinjection via the efferent duct and the subsequent electroporation of mouse testis. This procedure can be the means of choice to achieve fast, specific and efficient transfection of seminiferous tubules of mouse testis in vivo in order to investigate processes of spermatogenesis.

<table border="0" cellpadding="0" cellspacing="3">
	<tbody>
		<tr>
			<td>
			centrifuge
			</td>
			<td>Sigma</td>
			<td>1-15 PK</td>
			<td></td>
		</tr>
		<tr>
			<td>spectrophotometer</td>
			<td>Nanodrop</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>micropipette puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td>vertical capillary puller</td>
		</tr>
		<tr>
			<td>microinjection capillaries</td>
			<td>Clark</td>
			<td>GC100-10</td>
			<td>borosilicate standard wall</td>
		</tr>
		<tr>
			<td>micropipette beveler</td>
			<td>Bachofer</td>
			<td>Typ 462</td>
			<td>rotating disk beveler</td>
		</tr>
		<tr>
			<td>electroporator</td>
			<td>Nepagene</td>
			<td>CUY 21EDIT</td>
			<td>square wave electroporator</td>
		</tr>
		<tr>
			<td>tweezer electrodes</td>
			<td>Nepagene</td>
			<td>CUY 650P5</td>
			<td>5 mm &#216; disk electrodes</td>
		</tr>
		<tr>
			<td>stereo microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr>
			<td>cold light source</td>
			<td>Zeiss</td>
			<td>KL 1500LCD</td>
			<td></td>
		</tr>
		<tr>
			<td>microinjection pump</td>
			<td>Eppendorf</td>
			<td>Femtojet</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>homemade</td>
			<td></td>
			<td>XYZ cross table</td>
		</tr>
		<tr>
			<td>surgical instruments</td>
			<td>FST</td>
			<td></td>
			<td>scissors, forceps, 
			needle holder</td>
		</tr>
		<tr>
			<td>fine forceps</td>
			<td>FST</td>
			<td>Dumont #5, #7</td>
			<td>efferent ducts preparation</td>
		</tr>
		<tr>
			<td>Michel clip applying stapler</td>
			<td>FST</td>
			<td>Michel, 12029-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Michel suture clips</td>
			<td>FST</td>
			<td>Michel, 12040-01</td>
			<td>7.5 x 1.75 mm</td>
		</tr>
		<tr>
			<td>surgical suture</td>
			<td>Catgut 00</td>
			<td>Catgut 
			Markenkirchen</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe, with 30G needle</td>
			<td>B. Braun</td>
			<td>Omnican 40</td>
			<td>to load micropipette and 
			for anesthesia</td>
		</tr>
		<tr>
			<td>plasmid isolation kit</td>
			<td>Promega</td>
			<td>Cat. # A2495</td>
			<td>plasmid Midiprep</td>
		</tr>
		<tr>
			<td>plasmid pEGFP-C1</td>
			<td>Clontech</td>
			<td>Cat. #6084-1</td>
			<td>CMV-promoter + EGFP</td>
		</tr>
		<tr>
			<td>plasmid pDsRed2-N1</td>
			<td>Clontech</td>
			<td>Cat. #6084-1</td>
			<td>CMV-promoter + DsRed2</td>
		</tr>
		<tr>
			<td>Fast Green dye</td>
			<td>Sigma</td>
			<td>F7258-25G</td>
			<td>for dilution in ddH2O</td>
		</tr>
		<tr>
			<td>10x PBS, pH 7.4</td>
			<td>Carl Roth</td>
			<td>3957.1</td>
			<td>NaCl [1.37 M]</td>
		</tr>
		<tr>
			<td>6781.1</td>
			<td>KCl [27 mM]</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T106.2</td>
			<td>Na2HPO4+2H2O [100 mM]</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3904.1</td>
			<td>KH2PO4 [18 mM]</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% Ketamin</td>
			<td>Serum Werk 
			Bernburg</td>
			<td>Urotamin</td>
			<td>mix in a rate 1:1</td>
		</tr>
		<tr>
			<td>2% Xylazin</td>
			<td>Serum Werk 
			Bernburg</td>
			<td>Xylazin</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilium</td>
			<td>Bode Chemie</td>
			<td>Sterillium</td>
			<td>disinfection</td>
		</tr>
		<tr>
			<td>vet ointment</td>
			<td>S&#38;K Pharma 
			GmbH</td>
			<td>Kerato Biciron 5% 
			Augensalbe</td>
			<td>or similar to prevent 
			dryness of eyes</td>
		</tr>
		<tr>
			<td>To-Pro-3 iodide</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo microinjection and electroporation of mouse testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

The experimental setting for performing microinjection and electroporation of mouse testis in vivo as it is used according to the protocol is illustrated in Figure 1. Even though it is possible to acquire industrially manufactured micropipettes, we preferred to generate our own pipettes by pulling (Figure 1A) and beveling (Figure 1B) glass capillaries so that they fitted our needs. The equipment for microinjection and electroporation is illustrated in Fi...

Watch this Scientific Journal Video about In Vivo Microinjection and Electroporation of Mouse Testis at JoVE.com

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

in-vivo-microinjection-and-electroporation-of-mouse-testis

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JoVE Journal

Biology

27.7K Views.  Leibniz Institute for Farm Animal Biology (FBN). This article describes microinjection and electroporation of mouse testis in vivo as a transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

Video: In Vivo Microinjection and Electroporation of Mouse Testis

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

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Subtitles

Methods Article

In Vivo Microinjection and Electroporation of Mouse Testis