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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

In Vivo Microinjection and Electroporation of Mouse Testis

Published: August 23rd, 2014

DOI:

10.3791/51802

1Institute of Reproductive Biology, 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.

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 cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red 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.

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 qu....

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All performed animal experiments have been approved by the local ethics committee (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei, Mecklenburg-Vorpommern, Germany).

1. Plasmid Preparation

  1. 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 solutio.......

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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.......

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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 .......

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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). 

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NameCompanyCatalog NumberComments

centrifuge

Sigma1-15 PK
spectrophotometerNanodropND-1000
micropipette pullerNarishigePC-10vertical capillary puller
microinjection capillariesClarkGC100-10borosilicate standard wall
micropipette bevelerBachoferTyp 462rotating disk beveler
electroporatorNepageneCUY 21EDITsquare wave electroporator
tweezer electrodesNepageneCUY 650P55 mm Ø disk electrodes
stereo microscopeZeissStemi 2000-C
cold light sourceZeissKL 1500LCD
microinjection pumpEppendorfFemtojet
micromanipulatorhomemadeXYZ cross table
surgical instrumentsFSTscissors, forceps,
needle holder
fine forcepsFSTDumont #5, #7efferent ducts preparation
Michel clip applying staplerFSTMichel, 12029-12
Michel suture clipsFSTMichel, 12040-017.5 x 1.75 mm
surgical sutureCatgut 00Catgut
Markenkirchen
syringe, with 30G needleB. BraunOmnican 40to load micropipette and
for anesthesia
plasmid isolation kitPromegaCat. # A2495plasmid Midiprep
plasmid pEGFP-C1ClontechCat. #6084-1CMV-promoter + EGFP
plasmid pDsRed2-N1ClontechCat. #6084-1CMV-promoter + DsRed2
Fast Green dyeSigmaF7258-25Gfor dilution in ddH2O
10x PBS, pH 7.4Carl Roth3957.1NaCl [1.37 M]
6781.1KCl [27 mM]
T106.2Na2HPO4+2H2O [100 mM]
3904.1KH2PO4 [18 mM]
10% KetaminSerum Werk
Bernburg
Urotaminmix in a rate 1:1
2% XylazinSerum Werk
Bernburg
Xylazin
SteriliumBode ChemieSterilliumdisinfection
vet ointmentS&K Pharma
GmbH
Kerato Biciron 5%
Augensalbe
or similar to prevent
dryness of eyes
To-Pro-3 iodideInvitrogenT3605

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