Gene functional research using polyethylenimine-mediated in vivo gene transfection into mouse spermatogenic cells

Aim:To study polyethylenimine(PEI)-mediated in vivo gene transfection into testis cells and preliminary functionalresearch of spermatogenic cell-specific gene NYD-SP12 using this method.Methods:PEI/DNA complexes wereintroduced into the seminiferous tubules of mouse testes using intratesticular injection.Transfection efficiency andspeciality were analyzed on the third day of transfection with fluorescent microscopy and hematoxylin staining.Thelong-lasting expression of the GFP-NYD-SP12 fusion protein and its subcelluar localization in spermatogenic cells atdifferent stages were analyzed with fluorescent microscopy and propidium iodide staining.Results:With the media-tion of PEI,the GFP-NYD-SP12 fusion gene was efficiently transferred and expressed in the germ cells(especially inprimary spermatocytes).Transfection into Sertoli cells was not observed.The subcellular localization of the GFP-NYD-SP2 fusion protein showed dynamic shifts in spermatogenic cells at different stages during spermatogenesis.Conclusion:PEI can efficiently mediate gene transfer into spermatocytes.Thus,it might be useful for the functionalresearch of spermatogenic-cell specific genes such as the NYD-SP12 gene.In our study,the NYD-SP12 protein wasvisualized and was involved in the formation of acrosome during spermatogenesis.Our research will continue into thedetailed function of NYD-SP12 in spermatocytes.(Asian J Androl 2006 Jan;8:53-59)     

In vivo transfer of a neuronal nitric oxide synthase expression vector into the rat bladder by electroporation

OBJECTIVE: To investigate the possibility of in vivo gene transfer by attempting to transfer the neuronal nitric oxide synthase (nNOS) gene into rat bladder using electroporation. MATERIALS AND METHODS: The bladder was exposed through an abdominal midline incision in 8-week-old male rats. Plasmid DNA of the marker genes green fluorescent protein (GFP) and luciferase, and the nNOS gene, was then injected into the subserosal space of the bladder and electroporation applied. At 72 h after gene transfer, GFP and luciferase were assayed in the isolated bladder and immunohistochemical staining used to detect nNOS; NO(x) released from isolated bladder strips was also assessed using microdialysis and high-performance liquid chromatography. RESULTS: From the luciferase assay, 45 V, 1 Hz, 50 ms and eight pulses were selected as the optimum conditions for electroporation. Bladder specimens with GFP genes injected by electroporation showed bright and numerous sites of GFP expression in the smooth muscle layer. In rats with the nNOS gene injected by electroporation there was marked nNOS immunoreactivity, and NO(x) released from bladder strips was significantly greater than in the control groups. CONCLUSIONS: These results suggest that electroporation is a useful technique for in vivo gene transfer into rat bladder smooth muscles, and that the nNOS gene transferred by this procedure functionally expresses and contributes to NO production.     

Ex vivo and in vivo gene transfer to the peritoneal membrane in a rat model.

The efficacy of peritoneal dialysis (PD) depends on preserving the structural integrity and dialysing capacity of the peritoneal membrane. Membrane structure and function can change on PD, resulting in decreased membrane performance and possible discontinuation of PD as a treatment modality. We hypothesized that a gene therapy strategy might be an innovative and promising approach to maintaining membrane integrity and dialysing capacity in the PD population. We have characterized two methods of genetic modification of the peritoneal membrane in a rat model to test the feasibility of this approach. In ex vivo gene transfer, mesothelial cells are isolated from the peritoneal membrane, genetically modified in culture and subsequently re-implanted back onto the peritoneal membrane of syngeneic recipients. In in vivo gene transfer, genetic modification of the membrane is accomplished in situ through adenovirus-mediated delivery of the genetic material into the peritoneal cavity. The peritoneal membrane can be genetically modified to produce factors that may be of therapeutic value in maintaining the fibrinolytic balance in the peritoneal cavity, moderating peritoneal inflammation, and studying the development of peritoneal fibrosis. These models provide a basis for studying the contribution of specific molecules to peritoneal membrane physiology. Optimally, they will be paired with other PD-relevant model systems to understand peritoneal physiology, identify ways to prevent membrane damage, and maintain dialysing performance. While gene transfer can be used as a tool to understand the individual roles of factors or pathways in peritoneal membrane physiology, it can also be developed as a therapy platform for improving membrane characteristics and enhancing the therapy of peritoneal dialysis.     

Adenovirus-mediated gene transfer using ex vivo perfusion of the heart graft

A replication-deficient adenovirus was used for ex vivo gene transfer into rat heart grafts under conditions simulating clinical transplantation. The adenoviral vector, AdHCMVsp1LacZ, containing an expression cassette of Escherichiae coli lacZ, was used to perfuse heart grafts during cold ischemia before transplantation. Heart grafts were perfused with University of Wisconsin (UW) solution containing either 0 pfu, 5×10¹⁰ pfu, or 1×10¹¹ pfu of viral vector, and were preserved for either 2 or 4 h and then transplanted into syngeneic recipients. The animals were killed at 1, 7, and 14 days after transplantation. The infection rate was assessed by histochemical staining for -galactosidase. Using polymerase chain reaction (PCR), viral DNA presence was confirmed in every graft perfused with viral vectors. The protein production from the transfected gene was confirmed by a functional protein assay. An efficient gene transfer was achieved with an infection rate of 1%–1.5% for all cardiac myocytes, as assessed by 5-bromo-4-chloro-indolyl--d-galactopyranoside (X-gal) staining. All studies were negative in the control grafts. Gene expression persisted for at least 10 days after transplantation. We thus conclude that an efficient adenovirus-mediated gene transfection and expression of gene products can be achieved in ex vivo perfusion of the heart graft during cold preservation.     

In vivo analysis of germ cell apoptosis reveals the existence of stage-specific `social' control of germ cell death in the seminiferous epithelium

It has become clear in recent years that programmed cell death is regulated during development by signals from other cells. Nevertheless, compared to the `social' control of cell proliferation, relatively little is known about the `social' control of cell death in other systems. Since in a previous study we showed that induced germ cell apoptosis occurs at specific stages of the spermatogenic cycle, in this study we aimed to ascertain the existence of supracellular control of germ cell death during spermatogenesis. Therefore, the TUNEL technique has been used to analyse whether all of the different germ cell types are induced to die at these specific stages in animals injected intratesticularly with one of several inducers of apoptosis. Our findings suggest that all of the investigated agents trigger apoptosis in all the diverse progenies of germ cells existing at stages I, XII or XIV of the spermatogenic cycle. In contrast, at most other stages the number of apoptotic cells was similar to that found in control animals. These data are consistent with the existence of an intercellular control of germ cell death during spermatogenesis. We conclude that the seminiferous epithelium provides a suitable in vivo model to study the mechanisms underlying the `social' control of apoptosis.     

In vivo targeted gene transfer in skin by the use of laser-induced stress waves

BACKGROUND AND OBJECTIVES: Much interest has been shown in the use of lasers for nonviral targeted gene transfer, since the spatial characteristics of laser light are quite well defined. The aim of this study was to demonstrate in vivo gene transfer by the use of laser-induced stress waves (LISWs). STUDY DESIGN/MATERIALS AND METHODS: After reporter genes had been intradermally injected to rat skin in vivo, a laser target was placed on the gene-injected skin. LISWs were generated by the irradiation of an elastic laser target with 532-nm nanosecond laser pulses of a Q-switched Nd:YAG laser. RESULTS: Levels of luciferase activities for the skin exposed to LISWs were two orders of magnitude higher than those for the skin injected with naked DNA. Expressions of enhanced green fluorescent protein (EGFP) and beta-galactosidase were observed only in the area that was exposed to LISWs, and in addition, epidermal cells were selectively transfected. No major side effects were observed, and luciferase activity levels as high as 10(5) RLU per mg of protein were sustained even 5 days after gene transfer. CONCLUSION: Highly efficient and site-specific gene transfer can be achieved by applying a few pulses of nanosecond pulsed LISWs to rat skin in vivo.     

Gene transfer to the rat kidney in vivo and ex vivo using an adenovirus vector: factors influencing transgene expression.

BACKGROUND: The characteristics of adenovirus-mediated gene transfer into the kidney are not well examined. We studied the effects of contact time and temperature on adenovirus-mediated transgene expression in rat kidneys, using catheter-based in vivo gene transfer and a rat renal transplant model ex vivo. METHODS: An adenovirus vector containing the luciferase (Ad-Luc) or beta-galactosidase (Ad-LacZ) gene was introduced in vivo into the kidney via a renal artery catheter. Various contact times and temperatures were evaluated. Ex vivo, the renal graft was injected with Ad-Luc through the renal artery, chilled for 60 min and then transplanted. Luciferase expression was evaluated periodically by a non-invasive bioimaging system or histology. Cells expressing the LacZ gene were identified by immunoelectron microscopy. RESULTS: In in vivo gene transfer, successful transgene expression was achieved; however, its efficiency was independent of contact time or temperature. In ex vivo gene transfer, transgene expression in the renal graft peaked early and gradually decreased. Strong gene expression was observed in the recipients' livers. LacZ expression was detected in fibroblasts, parietal epithelial cells of Bowman's capsule, mesangial cells, podocytes and tubular cells. CONCLUSIONS: This study generated new information about in vivo and ex vivo gene transfer into the kidney, which would be useful for renal gene therapy.     

利用高通量基因芯片建立与分析单侧隐睾症睾丸基因表达谱

目的:利用高通量基因芯片技术分析单侧隐睾症睾丸组织中差异表达的基因。方法:抽提6例单侧隐睾症患者和3例正常生育男性患者睾丸组织中mRNA,反转录后,探针标记cDNA。使用高通量cDNA微阵列人类全基因表达谱基因芯片(45 034个基因)检测睾丸组织基因表达谱。差异表达基因在MAS系统中进行Path-way和GO分析。结果:Ratio>3.0或<0.33的数据项为差异表达的基因,单侧隐睾症患者睾丸生精组织中共检测到346个差异表达基因。其中上调基因60个,主要分布在1、15、5和19号染色体,集中在细胞周期、纤毛或鞭毛运动、DNA复制等聚类。下调基因286个,主要分布在1、19、16和11号染色体等,集中在精子发生和抗凋亡相关的基因聚类中。结论:单侧隐睾症涉及多功能基因表达的变化;单侧隐睾症睾丸基因表达谱的建立可为分析单侧隐睾症的遗传因素和探讨其生精功能异常的病因提供新的理论基础。     

B2 bradykinin receptor mediates the stimulatory effect of bradykinin on rat germ cell proliferation in vitro

The contribution of B1 and B2 bradykinin receptors to germ cell proliferation was studied by using in vitro organ cultures of testicular fragments of 3.5-day-old rats in the presence of 3H thymidine. Different combinations of agonists and antagonists of B1 and B2 receptors exerted differential mitogenic effect on pro-spermatogonial cells. Application of bradykinin (B2 receptor agonist) alone induced a threefold increase in germ cell labelling index compared with the control, whereas des-Arg9-bradykinin (B1 receptor agonist) caused weak stimulation (24%) on spermatogonial mitotic activity. The bradykinin-induced germ cell proliferation was significantly affected by B2 receptor antagonist (HOE-140) but not by B1 receptor antagonist. When B1 or B2 receptor antagonists were applied with des-Arg9-bradykinin, the germ cell labelling indices were nonsignificantly different compared with those of B1 receptor agonist only. The present findings suggest that B2 receptor is involved in mediating the stimulatory effect of bradykinin on germ cell proliferation and therefore bradykinin might be an important local testicular factor in the regulation of spermatogonial division and germ cell number.     

Evolution of somatic and germ cell populations after busulfan treatment in utero or neonatal cryptorchidism in the rat

Summary.  In order to elucidate the respective effects of depletion of germ cells and of increase in testicular temperature, rats of the same Wistar strain were rendered experimentally bicryptorchid or sterilized by a busulfan injection in utero and compared to control animals. In both models, germ cells were depleted but numeric evolution and functions of somatic cells differed. The aim of that work was to compare the numeric evolutions of testicular somatic and germ cells to their respective functions in each model before puberty and in adult rats of the same strain. Serum concentrations of FSH, LH and testosterone were compared at 20, 40 and 110 days of age. Histological analyses of Sertoli and germ cells in the seminiferous tubules and of Leydig cells in the intertubular tissue were performed before puberty and at adulthood. Testosterone serum concentrations were depleted in both models starting at 40 days of age and more in busulfan-treated rats. Both FSH and LH levels were increased from 20 days onwards in experimental rats. Additional cryptorchidism in busulfan-treated rats depressed the serum testosterone concentration. At 17 days of age, the cryptorchidism do not modify somatic or germ cell populations while busulfan treatment has induced a decrease of both these populations. Conversely, the cross sectional area of the somatic testicular cells was not affected whatever the treatment. In adult testes of busulfan-treated and cryptorchid rats, the total numbers of Sertoli and Leydig cells and of germ cells per testis were decreased. The cellular size of the perivascular Leydig cells was not modified by any of the treatments whereas the size of the Sertoli cells was reduced.
In conclusion, in both models the absence of germ cells induces a decrease in Sertoli cell function, while the increase in testicular temperature provokes degeneracies of Sertoli and germ cells in the seminiferous tubules of the rat.     

NucleofectorTM电转仪转染外源基因至骨髓基质细胞优化方案

目的用Nucleofector^TM电转仪介导真核表达载体pEGFP-C1及pEGFP-C1-PDX-1转染大鼠骨髓来源细胞，并对其转染条件进行优化以获得较高效率。方法将已构建好的重组载体以Nacleofector^TM电转仪转染不同方法获得的骨髓来源细胞，改变DNA的量、转染后血清浓度，在荧光显微镜下观察荧光并计算转染效率；转染后48h进行逆转录-聚合酶链反应（RT-PCR）检测目的基因的表达情况。结果相同条件下转染骨髓来源的神经干细胞（NSC）较转染骨髓基质细胞（MSC）可获得更高的转染效率；质粒2～10雌获得最佳的转染效率；提高转染后培养基的血清浓度可提高转染后细胞存活率及转染效率；RT-PCR检测转染后骨髓来源nestin＋细胞有PDX-1表达。结论通过优化转染条件提高了pEGFP-C1-PDX-1转染大鼠骨髓来源nestin＋细胞的效率，为成为组织工程的种子细胞提供了实验依据。     

腺病毒介导的NT-3基因在骨髓间质干细胞中的表达

目的:研究腺病毒介导的NT-3基因在培养的大鼠骨髓间质干细胞(mesenchymal stem cells,MSCs)中的表达.方法:在293细胞中培养扩增NT-3重组腺病毒(adenovirus vector for NT-3,Ad-NT-3),测定病毒滴度,然后用Ad-NT-3感染传代培养的MSCs,RT-PCR技术检测NT-3基因的表达.结果:Ad-NT-3扩增后获得了较高滴度的病毒,MSCs经Ad-NT-3感染后有NT-3 mRNA的转录.结论:腺病毒介导的NT-3基因可转入培养的MSCs并高效表达,为NT-3基因治疗的研究奠定了基础.     

Evaluation of the effect of selective germ cell depletion on subsequent spermatogenesis and fertility in the rat

Rats were treated with a single high dose of methoxy acetic acid (MAA; 650 mg/kg) specifically to deplete seminiferous tubules of pachytene and later spermatocytes. The impact of this selective depletion on subsequent spermatogenesis, sperm output and fertility was then evaluated at intervals ranging from 3 days to 10 weeks. Cauda epididymal sperm number was reduced progressively beyond 2 weeks post-treatment and reached a nadir at 5-6 weeks (28-34% of control values) before recovering progressively back to control levels at 10 weeks. Sperm motility was reduced significantly at 4-7 weeks post-treatment with a nadir at 6 weeks (35% of control values). Thus, at 5-6 weeks after MAA treatment, motile sperm output was reduced by 82-88%. Despite these changes, there was little evidence for infertility in the majority of treated males during a serial mating trial. Evaluation of seminiferous tubule morphology combined with germ cell counts at stage VII of the spermatogenic cycle confirmed that, initially, MAA induced the specific loss of pachytene and later spermatocytes at all stages other than early to mid stage VII. Maturation depletion of germ cells at later intervals was consistent with the initial effects of MAA, although at 21 days post-treatment a number of unpredicted (? secondary) changes in spermatogenesis were observed. These were (a) a reduction in number of pachytene spermatocytes at late stage VII/early stage VIII, (b) retention of sperm at stages IX-XIV, and (c) increased degeneration of pachytene spermatocytes and round spermatids at stage VII and of secondary spermatocytes at stages XIV-I. Whilst none of these changes was severe, together they probably accounted for the unexpectedly prolonged drop in sperm output. It is concluded that whilst deleterious changes in spermatogenesis may occur secondarily following MAA treatment, for the most part spermatogenesis proceeds normally and fertility is largely maintained despite a massive but transient decrease in sperm output.     

Total vascular exclusion safely facilitates liver specific gene transfer by the HVJ (sendai virus)-liposome method in rats

BACKGROUND: Most virus mediated transfection systems are efficient; however, their highly immunogenic properties do tend to cause clinical problems. HVJ-liposome vector is a hybrid vector consisting of liposome and inactivated sendai virus (hemagglutinating virus of Japan HVJ), which has been reported to be have a low immunogenicity, while it can also be repeatedly administered. To enhance the transfection efficiency, especially in the liver, we investigated the efficacy of total vascular exclusion (TVE) during the portal vein injection (PVI) of the vectors. MATERIALS AND METHODS: beta-galactosidase and luciferase expression were used as reporter genes. Wistar rats were injected with HVJ-liposome through PVI without TVE (PVI group, n = 10) or PVI with TVE (PVI + TVE group, n = 10). The control rats were infused with equal volumes of saline through the portal vein (control group n = 12). The transfection efficiencies were assessed by beta-galactosidase staining and a luciferase assay. Biochemical and histological analyses were performed to evaluate the tissue toxicity after gene transfer. RESULTS: The reporter genes expression in the liver dramatically increased after PVI + TVE in comparison to after PVI alone (1.2 x 10(5)versus 1.5 x 10(4) RLU/mg protein, P < 0.05 according to a luciferase assay). Notably, the extrahepatic "leaky" transgene expression could be minimized by PVI + TVE, whereas the general condition remained unchanged according to both the biochemical parameters and histological findings. CONCLUSIONS: The present data indicate that PVI + TVE may thus facilitate the liver-specific gene delivery using the HVJ-liposome method and this modality might also be applicable to other gene transfer systems.     

一氧化氮合酶基因表达对实验性大鼠隐睾生殖细胞凋亡的影响

目的　研究诱生型一氧化氮合酶 (iNOS)及其mRNA表达与实验性大鼠隐睾生殖细胞发育、凋亡的关系。方法　 (1)采用SD雄性健康大鼠 16只 ,日龄 2 2天时复制单侧隐睾模型。 (2 )采用生物素 dUTP/酶标亲和素测定法检测睾丸生殖细胞凋亡。 (3)采用免疫组织化学方法检测大鼠睾丸生殖细胞中iNOS基因表达。 (4 )采用原位杂交法检测大鼠生殖细胞中iNOSmRNA的表达。结果　 (1)术后第 7天 ,与自身对侧正常睾丸相比 ,隐睾侧睾丸发生凋亡的生殖细胞数显著增加 (P <0 .0 1)。 (2 )单侧隐睾模型建立术后第 7天 ,在双侧睾丸的间质细胞、支持细胞和初级精母细胞中均可见iNOS蛋白及iNOSmRNA的弱阳性表达 ,在隐睾侧睾丸曲细精管中脱落的生殖细胞中可见iNOS蛋白及iNOSmRNA的强阳性表达。术后第 7天 ,与自身对侧正常睾丸相比 ,隐睾侧睾丸生殖细胞中iNOSmRNA表达显著增加 (P <0 .0 1)。结论　 (1)实验性大鼠隐睾可以导致睾丸生殖细胞凋亡增加。 (2 )iNOS蛋白及其mRNA的表达增加是隐睾生殖细胞凋亡增加的分子机制之一