Erythropoietin gene transfer into rat testes by in vivo electropo-ration may reduce the risk of germ cell loss caused by cryptorchidism

Aim： To investigate the effects of rat Erythropoietin （Epo） on spermatogenesis by transferring rat Epo gene into cryptorchid testes by means of in vivo electroporation. Methods： Sprague-Dawley rats with surgically-induced unilateral cryptorchidism were divided into three groups： the first group was given intratesticular injections of pCAGGS-Epo （pCAGGS-Epo group）, the second group was given intratesticular injections of pCAGGS （pCAGGS group）, and the third group were given intratesticular injections of phosphate-buffered saline （PBS group）. At the same time, square electric pulses of 30 V were applied six times with a time constant of 100 ms. One or two weeks after injection, each testis was weighed and the ratio of the total number of germ cells to that of Sertoli cells （G/S ratio） was calculated to evaluate the impairment of spermatogenesis. Ten testes taken from each of the three groups were examined at each time point. Results： The testicular weight after the injection of pCAGGS-Epo or pCAGGS control plasmid was （0.85 ± 0.08） g and （0.83 ± 0.03） g, respectively, at week 1 （P = 0.788） and （0.62 ± 0.06） g and （0.52 ± 0.02） g, respectively, at week 2 （P = 0.047）. At week 1, spermatids and sperm were more abundant in testes with pCAGGS-Epo than those in the control testes. At week 2, spermatids and sperm were hardly detected in either group. The G/S ratio was 23.27 ± 6.80 vs. 18.63 ± 5.30 at week 1 （P = 0.0078） and 7.16 ± 3.06 vs. 6.05 ± 1.58 at week 2 （P = 0.1471）, respectively. Conclusion： The transfer of Epo to rat testes by in vivo electroporation may reduce the risk of the germ cell loss caused by cryptorchidism.

Intrastriatal Gene Transfer of Vascular Endothelial Growth Factor Rescues Dopaminergic Neurons in a Rat Parkinson's Disease Model

To examine the ability of intrastriatal gene transfer of vascular endothelial growth factor 165 mediated by adenoviral vector to rescue dopaminergic neurons in a rat model of Parkinson＇s disease （PD）, we constructed recombinant replication-deficent adenoviral vectors carrying the gene of VEGF165 （Ad-VEGF）, and injected Ad-VEGF （or Ad-LacZ and PBS as controls） into the striatum of rats 7 days after the lesion by 6-hydroxydopamine. The rat rotational behavior analysis and tyrosine hydroxylase （TH） immunohistochemistry were performed to assess the change of dopaminergic neurons. Our results showed that the rats receiving Ad-VEGF injection displayed a significant improvement in apomorphine-induced rotational behavior and a significant preservation of TH-positive neurons and fibers compared with control animals, It is concluded that intrastriatal gene transfer by Ad-VEGF may rescue the dopaminergic neurons from degeneration in a rat model of PD.

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 functional research of spermatogenic cell-specific gene NYD-SP12 using this method. Methods： PEI/DNA complexes were introduced into the seminiferous tubules of mouse testes using intratesticular injection. Transfection efficiency and speciality were analyzed on the third day of transfection with fluorescent microscopy and hematoxylin staining. The long-lasting expression of the GFP-NYD-SP12 fusion protein and its subcelluar localization in spermatogenic cells at different stages were analyzed with fluorescent microscopy and propidium iodide staining. Results： With the mediation of PEI, the GFP-NYD-SP12 fusion gene was efficiently transferred and expressed in the germ cells （especially in primary 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 functional research of spermatogenic-cell specific genes such as the NYD-SP12 gene. In our gtudy, the NYD-SP12 protein was visualized and was involved in the formation of acrosome during spermatogenesis. Our research will continue into the detailed function of NYD-SP12 in spermatocytes. （Asian J Androl 2006 Jan; 8： 53-59）

Transient Expression of BVDV N^(pro) Gene in vitro and Distribution of Fusion Protein in Cells

The gene encoding nonstructural protein Npro of BVDV was amplified by PCR,then was inserted into pEGFP-C1 vector.The 293 cells were transfected in vitro with recombinant plasmid by liposome.Npro-EGFP fusion protein was viewed directly with fluorensce microscope and mRNA expression of Npro was detected by reverse transcription-polymerase chain reaction（RT-PCR）.Results showed that recombinant plasmid was confirmed to be constructed correctly by PCR,restriction enzyme digestion and DNA sequencing;meanwhile,the gene carried was expressed in 293 cells.The fusion protein had properties of both the two Npro and enhanced green fluorescent proteins（EGFP） and distributed in the cytoplasm.This research lays a foundation for further researches which explored Npro gene function in the process of viral replication and synthesis.

BPI_(700)-Fc∴1_(700) chimeric gene expression and its protective effect in a mice model of the lethal E. coli infection

Background Infections caused by gram-negative bacteria （GNB） often lead to high mortality in common clinical settings. The effect of traditional antibiotic therapy is hindered by drug-resistant bacteria and unneutralizable endotoxin. Few effective methods can protect high risk patients from bacterial infection. This study explored the protection of adeno-associated virus 2 （AAV2）-bacteriacidal permeability increasing protein 700 （BPI700） -fragment crystallizable gamma one 700 （Fcγ1700） chimeric gene transferred mice against the minimal lethal dose （MLD） of E.coli and application of gene therapy for bacterial infection. Methods After AAV2-BPI700-Fcγ1700 virus transfection, dot blotting and Western blotting were used to detect the target gene products in Chinese hamster ovary-K1 cells （CHO-Klcells）. Reverse transcription-polymerase chain reaction and immunohistochemical assay were carried out to show the target gene expression in mice. Modified BPI-enzyme linked immunosorbent assay was used to identify the target gene products in murine serum. The protection of BPI700-Fcγ1700 gene transferred mice was examined by survival rate after MLD E. coli challenge. Colony forming unit （CFU） count, limulus amebocyte lysate kit and cytokine kit were used to quantify the bacteria, the level of endotoxin, and proinflammatory cytokine. Results BPI1-99-Fcγ1 protein was identified in the CHO-K1 cell culture supernatant, injected muscles and serum of the gene transferred mice. After MLD E. coli challenge, the survival rate of AAV2-BPI700-Fcγ1700 gene transferred mice （36.7%） was significantly higher than that of AAV2-enhanced green fluorescent protein （AAV2- EGFP） gene transferred mice （3.3%） and PBS control mice （5.6%）. The survival rate of AAV2-BPI700-Fcγ1700 gene transferred mice treated with cefuroxime sodium was 65.0%. The bacterium number in main viscera, the levels of endotoxin and proinflammatory cytokine （tumor necrcsis factor-α and interleukin-1β） in serum of the AAV2-BPI700-Fcγ1700 gene transferred mice were markedly lower than that of PBS control mice （P〈0.01）. Conclusions AAV2-BPI700-Fcγ1700 gene transferred mice can resist MLD E. coli infection through expressing BPI1-199-Fcγ1 protein. Our findings suggested that AAV2 mediated BPI700-Fcγ1700 gene delivery could be used for protection and treatment of clinical GNB infection in high-risk individuals.