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Research Article |
Department of Pharmacology,4 University of Oxford, Oxford OX1 3QT, United Kingdom
Department of Urology,5 Nagoya City University, Nagoya 467-8601, Japan
Royal Veterinary College,6 London NW1 OUT, United Kingdom
Institute of Zoology,7 The Zoological Society of London, London NW1 4RY, United Kingdom
ABSTRACT
The study of gene function in testis and sperm has been greatly assisted by transgenic mouse models. Recently, an alternative way of expressing transgenes in mouse testis has been developed that uses electroporation to introduce transgenes into the male germ cells. This approach has been successfully used to transiently express reporter genes driven by constitutive and testis-specific promoters. It has been proposed as an alternative method for studying gene function in testis and sperm, and as a novel way to create transgenic animals. However, the low levels and transient nature of transgene expression that can be achieved using this technique have raised concerns about its practical usefulness. It has also not been demonstrated in mammals other than mice. In this study, we show for the first time that in vivo gene transfer using electroporation can be used to express a fluorescent transgene in the testis of a mammal other than mice, the Syrian golden hamster. Significantly, for the first time we demonstrate expression of a transgene in epididymal sperm using this approach. We show that expression of the transgene can be detected in sperm for as long as 60 days following gene transfer. Finally, we provide the first systematic demonstration that this technique does not lead to any significant long-term adverse effects on testicular integrity and sperm quality. This technique therefore offers a novel way to study gene function during fertilization in hamsters and may also have potential as a way of creating transgenic versions of this important model species.
electroporation, epididymis, gamete biology, in vivo gene transfer, sperm, spermatogenesis, testis
INTRODUCTION
The creation of transgenic mice has been one of the key developments in the study of gene function in the living organism. Transgenic mice are generally created by injection of transgenes into the pronucleus of a fertilized egg [1]. Transgenic approaches have been particularly important for studying gene function in the testis and sperm [2, 3] because of the current lack of a way of fully recapitulating spermatogenesis in the culture dish [4]. However, the production of transgenic mice remains a costly and laborious process. In addition, it has not been possible to use this approach to create a transgenic version of another mammalian species, such as the hamster, that has been used extensively for the study of reproduction [5].
Recently an alternative way to study gene expression in the testis has been pioneered that involves in vivo gene transfer of transgenes into testicular cells by electroporation. A number of studies in mice have shown that it is possible to use this approach to express reporter genes such as lacZ, and green fluorescent protein (GFP) and its variants, under the control of constitutive or testis-specific gene promoters, in spermatogenic cells [6–11], and in one case in testicular sperm [8]. In this latter case, the testicular sperm were dissected from the testis and used to create transgenic offspring by intracytoplasmic sperm injection (ICSI) [8].
These findings have led to the proposal that in vivo gene transfer into the testis represents an alternative way to study gene expression in testis and sperm, as well as a potential new way of creating transgenic animals. However, doubts have been raised about the usefulness of the technique as a practical alternative to a standard transgenic approach [12]. One problem is that although a high level of expression of the transgene can be detected after 24–48 h following the electroporation procedure, at later times the level of expression in the male germ cells appears to drop substantially [6–12]. Significantly, there have been no reports of expression of transgenes in epididymal sperm after in vivo gene transfer into the testis by electroporation. Finally, although expression of transgenes in the testis has been demonstrated in mice using this approach, it is unclear how applicable this approach is to other mammalian species important for studies of fertilization and other aspects of reproduction, such as the hamster.
In this study, we have looked to see whether in vivo gene transfer into the testis by electroporation can be applied to mammals other than mice. We initially chose to study the Syrian golden hamster because of the important role that this species has played in studies of mammalian egg activation [13–16]. The hamster is also the chosen organism for a number of reproductive research models including the reproductive endocrinology of embryo implantation [17–18], reproductive aging [19], endocrine disruption [20] and the effects of smoking/nicotine upon oviductal function [21]. Another key aim of this study was to see whether in vivo gene transfer into the testis by electroporation can be used to generate epididymal sperm expressing a transgene, as this could have major importance for studies of important physiological processes such as the acrosome reaction, sperm-egg binding and fusion, and egg activation. Finally, we have also undertaken the first systematic study to see what negative effects, if any, the in vivo gene transfer procedure has upon important indicators of testicular integrity and sperm quality.
MATERIALS AND METHODS
Animals
Adult male Golden Syrian hamsters (4–6 wk of age) were obtained from Harlan UK Ltd. Animals were maintained on a 12L:12D photoperiod and provided with food and water ad libitum. All procedures described herein conform to the Animals (Scientific Procedures) Act 1986.
Expression Vector
A mammalian expression construct (EYFP-Mito), designed to produce enhanced yellow fluorescent protein (EYFP), was obtained from Professor Norio Nakatsuji, (Kyoto University, Kyoto, Japan). This protein expression vector contained the promoter enhancer of human cytomegalovirus, the promoter of chicken ß-actin, a ß-actin intron, and the rabbit ß-globin polyadenylation signal. The vector also contained a mitochondrial localization signal, which leads to the sequestration of the EYFP into mitochondria as described previously [8]. For in vivo gene transfer by electroporation, EYFP-Mito was precipitated overnight and resuspended in an appropriate volume of Hepes-buffered saline (HBS) to obtain a 3 µg/µl working solution, with trypan blue (Sigma) added to a final concentration of 0.04% to monitor micropipette position and injection accuracy/volume during gene transfer.
In Vivo Gene Transfer by Electroporation
Animals were anesthetized with appropriate proportions of medical grade oxygen and Isoflurane (Animal Care Ltd). Animals were provided with analgesia perioperatively (4 mg/kg Rimadyl; Pfizer Ltd) by subcutaneous injection. Eye protection was provided by applying an appropriate amount of liquid gel carbomer (Viscotears; Novartis) before surgery. For each animal, the right testis was treated as the experimental organ and the left testis remained completely untouched throughout the procedure to act as a control. Fur was removed from the lower right abdominal quadrant and a small incision made in the skin and muscle layer to allow the right testis to be exposed and removed from the body cavity. The efferent duct was located at the proximal apex of the testis and EYFP-Mito injected by gentle mouth pressure into the rete testis via a sharpened glass microcapillary pipette. EYFP-Mito was injected until the majority (80%) of the seminiferous tubules visible on the surface of the testis displayed trypan blue (typically 70–90 µl, 210–270 µg). An electrical current was then applied to the right testis using tweezer-type electrodes linked to an Electrosquare Porator ECM830 (BTX) using a voltage of 50 V and pulse length of 50 msec. The testis was replaced, the muscle and skin layers sutured, and the animal allowed to recover. Animals were maintained for varying periods of time (20–60 days) before they were killed, depending on experimental requirement.
Analysis of EYFP-Mito Expression
The right testes of 12 hamsters were injected with EYFP-Mito and electroporated as described earlier. Animals were subsequently killed at 20 days (n = 4), 40 days (n = 4) and 60 days (n = 4) postsurgery. Testes (left control and right experimental) from the animals killed on Day 20 were teased apart in PBS and individual seminiferous tubules transferred onto an agar stage and analyzed for expression of EYFP-Mito on a scanning confocal microscope (Leica) set to an appropriate wavelength for YFP. Animals killed on either Day 40 or Day 60 postsurgery were treated such that both thin histological sections and mature epididymal sperm could be analyzed for expression of EYFP-Mito. The epididymis was separated from each testis and the testis snap frozen in liquid nitrogen to await histological analysis. Epididymal sperm were obtained by partially macerating the epididymis in PBS and allowing sperm to swim free from the surrounding tissue. Epididymal sperm were then counterstained with propidium iodide (1 µg/µl; Sigma) and analyzed using a fluorescence microscope (Leica) equipped with an excitation light source and Texas Red (for propidium iodide staining) and YFP filter sets. Images were captured using Leica IM50 Image Manager software. Serial frozen sections (10 µm in thickness) were prepared from frozen testis (at 100 µm intervals), transferred onto glass slides coated with 0.1% w/v poly-L-lysine (Sigma), fixed for 20 min in Bouin's solution (Sigma, UK), counterstained with neutral red (0.01% w/v neutral red, 0.01% w/v sodium azide, 0.04% v/v glacial acetic acid) and finally analyzed for expression of EYFP-Mito using a fluorescence microscope as described earlier.
Analysis of Testicular Integrity
To investigate the potential effect of in vivo gene transfer by electroporation upon testicular health in the hamster, a second group of animals (n = 20) was divided into four experimental groups (labeled 1–4, n = 5 per group) and subjected to the assigned experimental intervention and maintained postprocedure for a total period of 40 days. Experimental intervention assigned to each group was as follows: group 1, testis removed from peritoneal cavity and exposed for 5 min; group 2, testis removed from peritoneal cavity and rete testis injected with 80–100 µl of vehicle (HBS containing 0.04% trypan blue); group 3, testis removed from peritoneal cavity and rete testis injected with 80–100 µl of vehicle (HBS containing 0.04% trypan blue) followed by electroporation (50 V, 50 msec), group 4, testis removed from peritoneal cavity and electroporated (50 V, 50 msec) without injection. After animals were killed, each testis was separated from its epididymis and snap frozen in liquid nitrogen. From each testis, 10 thin histological sections (10 µm), prepared as described above, were chosen at random for analysis of in situ cell death (apoptosis) using a TUNEL fluorescein-based kit (Roche Bioscience). For TUNEL analysis, histological sections were fixed in 4% paraformaldehyde, stained in accordance with the manufacturer's instructions, and analyzed by fluorescence microscopy using a GFP filter set. For each testis, two parameters were quantified: first, the number of seminiferous tubules containing TUNEL-positive cells and secondly, the total number of TUNEL-positive cells per tubule were recorded. TUNEL analysis involved a minimum of 200 tubules from at least 10 randomly selected sections. A further 10 randomly selected sections were fixed in 4% glutaraldehyde in PBS for 15 min and stained with hematoxylin and eosin. These sections were used to analyze spermatogenesis in a minimum of 200 tubules per testis. Tubules were classified into three basic groupings dependent upon the dominant population of cells in each histological section: 1) spermatogonia, 2) spermatocytes, and 3) round and elongating spermatids. These three classifications were designed to serve as a general system to analyze spermatogenesis in our experimental and control testes because they represent the three major stages of the spermatogenic process. Stages of spermatogenic cell development were identified in accordance with an earlier study [22].
Analysis of Sperm Viability
To investigate possible effects upon sperm viability, the cauda epididymis, obtained from killed animals in groups 1–4, was placed in 1 ml of prewarmed (37°C) BWW medium (100 mM NaCl, 2.8 mM KCl, 0.5 mM NaH2PO4, 2.0 mM CaCl, 0.5 mM MgCl, 25 mM NaHCO3, 10 mM HEPES, 5.0 mM glucose, 1.0 mM sodium pyruvate, 20 mM sodium lactate, 4 mg/ml w/v BSA). The tissue was partially macerated with a sterile scalpel and incubated at 37°C for 3 min to allow the sperm to swim free of the surrounding tissue. Sperm from each cauda epididymis were assessed for viability using a live to dead count ratio. After the sperm had been incubated for 3 min, 100 µl of sperm were mixed with 9 mM 5-carboxyfluorescein diacetate (CFDA; Sigma) and 24µM of the nuclear stain propidium iodide (Sigma). After 5 min incubation, samples were analyzed using fluorescence microscopy. A minimum of 200 sperm were counted per epididymis and the number of live sperm versus dead sperm was determined.
Analysis of Sperm Motility
Sperm samples from the left and right epididymis taken from killed animals in groups 1–4 were diluted to an appropriate volume to observe 15 cells at all times at 40x magnification on a negative high phase-contrast microscope (Olympus-BH2). Spermatozoa were recorded for 3 min per sample on a heated (37.5°C) stage using a monochrome video camera (Sony Hyper HAD) attached to the microscope. Approximately 10 µl of diluted sperm from each epididymis was placed on a glass slide, and a coverslip was positioned on top of the sample with a 100µm space as described by an earlier study [23]. Sperm motility was analyzed using a Hobson Sperm Tracker (Sheffield). A total of 100 spermatozoa were tracked in each sample and only if they remained within the calibration frame for 4 seconds; search radius was set to 18.75 µm. Analysis was in terms of three standard sperm motility characteristics: straightline velocity, average path velocity, and curvilinear velocity.
Statistical Analysis
Sperm viability (live versus dead) and motility (Hobson Sperm Tracker) data were arcsine transformed and analyzed using Minitab version 13.0. Cell viability and motility were compared between treatment groups using a nonparametric Kruskal-Wallis test. Cell viability and motility were compared between the left and right testis of each animal using a Wilcoxon test. For the assessment of spermatogenesis and testicular integrity, because the left testis in each animal was deemed to be the control for each intervention, all outcomes were calculated by subtracting the right testis values from those of the left. This meant that group means already took into account the control condition for each animal. This data were analyzed in SPSS using one-way ANOVA and the least significant difference post hoc test. In all statistical tests, a value of P 
0.05 was considered significant.
RESULTS
Expression of a Fluorescent Transgene in Hamster Testis Following In Vivo Gene Transfer by Electroporation
First we looked to see whether a transgene could be expressed in hamster testis and epididymal sperm following in vivo gene transfer by electroporation. We injected the right testis of hamsters with a DNA construct that expresses EYFP-Mito protein under the control of a constitutive promoter, and then used electroporation to drive the DNA into the cells of the testis. The DNA construct was injected into the rete testis rather than directly into the testis because this route of injection results in a substantially better level of expression of the transgene [7, 11]. Animals treated in this way were killed at 20, 40, and 60 days following the procedure, and the right, treated testis of each animal was analyzed for the presence of cells expressing EYFP-Mito protein and compared against the left control testis.
We found that 20 days after the procedure, seminiferous tubules of testes experiencing electroporation consistently exhibited clusters of fluorescent cells that were not evident in tubules from the control testis (Fig. 1, A and B). Histological sections of testis demonstrated that these fluorescent cells were mostly situated adjacent to the basement membrane of the seminiferous tubules (Fig. 1, C and D). This pattern of expression suggested that at this time point the transgene was primarily being expressed in the youngest male germ cells, the spermatogonia, because these are situated around the perimeter of the tubule. The discrete pattern of expression did not suggest that the transgene was being expressed in the Sertoli cells, because these have an elongated shape and tend to span the tubule from perimeter to lumen, producing a characteristic tree-shaped appearance.
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At 40 days after the procedure, discrete cells expressing EYFP fluorescence were clearly evident in thin histological sections of all testes electroporated with EYFP-Mito (Fig. 2, A and E) but not in control tissues (Fig. 2, B and F). EYFP-positive cells were generally located in small clusters within the epithelium of the seminiferous tubules. The size and shape of these positive cells suggested that they were most likely to be developing spermatogonia or spermatocytes (Fig. 2, A, C, E, and G).
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Even at 60 days, it was clear that discrete cells expressing EYFP fluorescence were still evident in the spermatogenic epithelium of electroporated testes previously electroporated with EYFP-Mito (Fig. 2, C and G), though in reduced proportions compared to those seen at 40 days following the procedure. These EYFP-positive cells, likely to be spermatogonia and spermatocytes, were not detectable in control tissues (Fig. 2, D and H).
Expression of a Fluorescent Transgene in Hamster Epididymal Sperm after In Vivo Gene Transfer
We next looked to see whether in vivo gene transfer into the testis by electroporation results in expression of the EYFP-Mito transgene in sperm. Previous studies using this approach in mice have not detected expression of transgenes in epididymal sperm, although one study did show expression in a few clumps of testicular sperm [8]. However, when we looked at sperm from the right testis at 40 days following the procedure, we saw epididymal sperm expressing EYFP fluorescence in their midpiece (Fig. 3, A and E), the expected site of localization of EYFP-Mito in sperm because it is here that the mitochondria are concentrated. This pattern of fluorescence was not evident in sperm from the control epididymis (Fig. 3, B and F). We estimated that approximately 10% of epididymal sperm examined appeared to exhibit EYFP fluorescence in their midpieces.
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At 60 days following the procedure, a proportion (preliminary estimations suggest this to be in the region of 5–10%) of sperm from the right epididymis also clearly expressed EYFP fluorescence in the midpiece (Fig. 3, C and G), and, as before, there was no EYFP fluorescence in sperm collected from the control epididymis (Fig. 3, D and H).
Effect of In Vivo Gene Transfer on Testicular Integrity and Spermatogenic Cycle
The question of whether in vivo gene transfer into the testis by electroporation has any detrimental effects upon testicular integrity and the spermatogenic cycle is an important one to address if this approach is to be used to study sperm function or to create transgenic animals. We partially addressed this question recently in mice when we investigated the potential effect of two different in vivo gene transfer techniques upon testicular cells [11]. In the present study, we looked at the effect in vivo gene transfer by electroporation had upon testicular cells in hamsters, as well as carrying out a far more systematic survey of the effects of the electroporation upon testicular integrity and the spermatogenic cycle than in our previous study.
First, we assigned animals to one of four experimental groups. Our aim was to isolate all the different variables involved in the in vivo gene transfer procedure and assess their potential negative effect upon testicular integrity independently. The experimental interventions in the different groups were as follows: 1) the testis was merely removed from the body cavity; 2) the testis was removed and injected with a vehicle solution; 3) the testis was removed and injected with a vehicle solution and electroporated; 4) the testis was removed and electroporated without injection. Animals were left for 40 days following the procedure, which should allow, under normal circumstances, for a complete renewal of the spermatogenic cells in the testis; the spermatogenic cycle in the hamster is estimated to take 35 days [24].
In each case, we looked to see what negative effect any of these interventions had on testicular cells by looking at the incidence of apoptotic cell death following the procedure. We studied two measured indicators of apoptotic cell death: first, the percentage of seminiferous tubules staining positively for apoptosis using the TUNEL kit; and second, the mean number of cells per tubule that were TUNEL-positive (apoptotic). Statistical analysis did not reveal any significant differences between the experimental groupings except for the percentage of tubules exhibiting TUNEL-positive cells (P < 0.05). Post hoc analysis showed that group 2 (injection of vehicle only) exhibited a significantly higher proportion of tubules containing TUNEL-positive cells than group 4 (electroporation only, P = 0.01) but not groups 1 or 3.
We next looked at the question of whether the spermatogenic cycle was affected adversely by the in vivo gene transfer procedure. Histological analysis of fixed testis sections stained between groups) of the differences between left (control) and right (electroporated) testis revealed no significant differences (P 
0.05) in the proportions of the different spermatogenic cell types (spermatogonia, spermatocytes, and round and elongating spermatids) between any of the animal groups or between the left and right testes (Fig. 4). Thus, the in vivo gene transfer procedure appears to have no significant effect on the rate of spermatogenesis or on the progression of the spermatogenic wave.
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Effect of In Vivo Gene Transfer on Sperm Viability and Motility
We next looked to see whether the in vivo gene transfer procedure had any detrimental effects upon sperm quality. Using the same four experimental groups, we looked at the effect of different aspects of the procedure upon two important measures of sperm quality: viability and motility. Sperm viability was assessed by propidium iodide staining, which is a nuclear stain and thus only stains sperm with compromised membranes. Sperm mobility was assessed using a Hobson Sperm Tracker. Three different motility characteristics were studied: curvilinear velocity, average path velocity and straight line velocity. Statistical analysis revealed no significant differences between samples of sperm obtained from the left (control) and the right (electroporated) epididymis of experimental animals, or between any of the four treatment groups (Fig. 5). Thus, it appears that sperm quality as measured by a number of different parameters is unaffected by the in vivo gene transfer procedure.
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DISCUSSION
In vivo gene transfer into the testis by electroporation has been put forward as an alternative way of studying testicular and sperm function and as a novel way of creating transgenic animals, and a number of studies in mice have demonstrated its potential [6–11]. However, so far experiments have failed to produce mature transgenic sperm, and it has yet to be demonstrated whether this approach can be used to express transgenes in the testis of other mammals. Here we have shown for the first time that in vivo gene transfer into the testis by electroporation can be used to achieve long-lived, high-level expression of a fluorescent transgene in the male germ cells of the Syrian golden hamster. We have also shown for the first time that this approach can be used to generate mature epididymal sperm expressing a fluorescent transgene. In this respect we have gone further than previous studies that observed expression of transgenes only in spermatogenic cells [6–11], or in one case, in testicular sperm [8]. Our success in this respect might be attributable to our decision to inject DNA expression constructs directly into the rete testis rather than simply into the testis or by intratubular injection. It is also conceivable that the hamster testis may be more responsive to this methodology than mouse testis. Our demonstration that this approach can be used to express transgenes in hamster testis is highly significant given the important role that this species has played historically in studies of spermatogenesis and fertilization [5]. The lack of a means of creating transgenic hamsters has limited studies of gene function during spermatogenesis and fertilization in this species, but our findings suggest that it should be possible to use in vivo gene transfer into the testis to study the role of genes in mediating important aspects of sperm development and function in this species. We have also shown that expression of the transgene can be detected in epididymal sperm, the first time that this has been demonstrated in any species using this approach. Our ability to generate hamster epididymal sperm expressing a fluorescent transgene suggests that it should be possible to also express fluorescently tagged recombinant versions of endogenous sperm proteins, for instance by fusing a sperm protein of interest to a fluorescent reporter protein such as GFP or one of its variants [25]. Such an approach has a number of advantages over immunocytochemistry in that changes in the pattern of localization could be followed in real time during important processes in the sperm such as capacitation, the acrosome reaction, sperm-egg binding and fusion, and even into the postfertilized egg.
Our findings also suggest that it may be possible to express dominant negative mutant versions of endogenous sperm proteins and observe their effect on these processes. Such strategies could potentially be employed in hamsters, but also in mice as an alternative to the standard transgenic approach.
If in vivo gene transfer into the testis is to be used to study gene function during spermatogenesis, it is important that the procedure does not itself disrupt normal testicular function. Our findings here represent the first systematic survey of whether the in vivo gene transfer procedure has detrimental effects upon testicular integrity and the spermatogenic cycle. This study goes further than our previous study that looked at this question in mice [11] in terms of number of parameters tested and the degree of quantitation. The fact that we found no significant differences in terms of apoptotic cell death or the pattern of spermatogenesis is important because it suggests that this approach can be used to study the mechanisms underlying the regulation of this process in a context where the procedure itself is not disrupting spermatogenesis. Further studies will be needed to confirm that more subtle changes, for instance the pattern of expression of key genes involved in spermatogenesis, are not altered following the in vivo gene transfer procedure.
The fact that we also show the in vivo gene transfer procedure appears to have no detrimental effects upon either sperm motility or sperm viability is also important from the point of view of creating transgenic animals. Our findings suggest that in vivo gene transfer into the testis by electroporation can be carried out without disrupting normal sperm function in the process. This suggests that sperm carrying transgenes generated by in vivo gene transfer could potentially be used to create transgenic hamsters.
The creation of a transgenic hamster would be highly important for a number of reasons. Aside from its importance as a model organism for reproductive research [13–21, 26], the hamster is also a key model organism in the study of energy metabolism, appetite regulation, and the control of adiposity [27–29] and as a model for human pancreatic cancer [30], carbohydrate-induced insulin resistance [31], certain types of muscular dystrophy [32], and cardiac dysfunction [33]. Development of a way of creating transgenic hamsters could have a major impact on these areas of research. The fact that we have detected expression of a transgene in epididymal sperm as late as 60 days after in vivo gene transfer suggests that the transgene may have integrated into the sperm genomic DNA, because the hamster spermatogenic cycle is approximately 35 days. If this is the case, then fertilization of hamster oocytes with these sperm could be used to generate transgenic offspring. Such integration should not be necessary for the use of in vivo gene transfer into the testis by electroporation as a transient method for assessing gene function in the testis and sperm. However, from the point of view of using this approach to create transgenic animals, it will be of great importance for future work to determine whether such integration into the sperm genome has indeed taken place, and indeed whether transgenes introduced into the sperm using this approach can be passed down to subsequent generations.
A previous study in mice created transgenic offspring by selecting testicular sperm expressing a transgene from animals that had undergone in vivo gene transfer into the testis by electroporation, and then using ICSI to fertilize hamster oocytes in vitro [8]. However, the fact that we observe an estimated 10% of sperm expressing a fluorescent transgene after in vivo gene transfer means that it may be possible to create transgenic offspring simply by natural mating of female hamsters with males that have undergone in vivo gene transfer in both testes. The predictable manner in which female hamsters undergo estrus and the ease with which hamsters can be bred [5] are valuable considerations in the use of this species for such studies. An alternative approach would be to use a dual expression construct in which a transgene of interest driven by its own promoter is present in the same vector as a fluorescent marker gene under the control of a testis specific promoter. Fluorescent sperm carrying the transgene could then be selected by fluorescence activated cell sorting and used to artificially inseminate females. The two genes would probably need to be separated by an insulator sequence to allow them to be expressed independently. Neither of these approaches would require the use of in vitro fertilization, an important advantage in species like the hamster in which manipulation and maintenance of the embryo have proven far from trivial [5]. We are currently exploring whether either of these two approaches can be used to generate transgenic hamsters.
ACKNOWLEDGMENTS
The authors would like to thank Professor Norio Nakatsuji (Kyoto University, Japan) for providing the EYFP-Mito construct used in this study.
FOOTNOTES
1 Supported by a Medical Research Council Non-Clinical Senior Fellowship awarded to J.P. 
2 Correpondence: John Parrington, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford. OX1 3QT, United Kingdom. FAX: 44 1865 270853; john.parrington{at}pharm.ox.ac.uk
3 These authors contributed equally to this work.
Received: 23 March 2005.
First decision: 6 May 2005.
Accepted: 12 September 2005.
REFERENCES
ciegli
ska D, Vydra N, Malusecka E, Krawczyk Z, In vivo electroporation of the testis versus transgenic mice model in functional studies of spermatocyte-specific hst70 gene promoter: a comparative study. Mol Reprod Dev 2003 65:382-388[CrossRef][Medline]This article has been cited by other articles:
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  M. Kanatsu-Shinohara, T. Muneto, J. Lee, M. Takenaka, S. Chuma, N. Nakatsuji, T. Horiuchi, and T. Shinohara Long-Term Culture of Male Germline Stem Cells From Hamster Testes Biol Reprod, April 1, 2008; 78(4): 611 - 617. [Abstract] [Full Text] [PDF]
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