scholarly journals Intra-testicular injection of adenoviral constructs results in Sertoli cell-specific gene expression and disruption of the seminiferous epithelium

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 361-370 ◽  
Author(s):  
R P Hooley ◽  
M Paterson ◽  
P Brown ◽  
K Kerr ◽  
P T K Saunders
Keyword(s):  
Seminiferous Tubule ◽  
Fluorescent Protein ◽  
Immune Cell ◽  
Adenoviral Vectors ◽  
Seminiferous Tubules ◽  
Specific Gene ◽  
Viral Particles ◽  
Junctional Complexes ◽  
Testicular Injection

Spermatogenesis is a complex process that cannot be modelledin vitro. The somatic Sertoli cells (SCs) within the seminiferous tubules perform a key role in supporting maturation of germ cells (GCs). Progress has been made in determining what aspects of SC function are critical to maintenance of fertility by developing rodent models based on the Cre/LoxP system; however, this is time-consuming and is only applicable to mice. The aim of the present study was to establish methods for direct injection of adenoviral vectors containing shRNA constructs into the testis as a way of inducing target-selective knock-downin vivo. We describe here a series of experiments using adenovirus expressing a green fluorescent protein (GFP) transgene. Injection via the efferent ductules resulted in SC-specific expression of GFP; expression levels paralleled the amount of infective viral particles injected. At the highest doses of virus seminiferous tubule architecture were grossly disturbed and immune cell invasion noted. At lower concentrations, the expression of GFP was variable/negligible, the seminiferous tubule lumen was maintained but stage-dependent GC loss and development of numerous basal vacuoles was observed. These resembled intercellular dilations of SC junctional complexes previously described in rats and may be a consequence of disturbances in SC function due to interaction of the viral particles with the coxsackie/adenovirus receptor that is a component of the junctional complexes within the blood testis barrier. In conclusion, intra-testicular injection of adenoviral vectors disturbs SC functionin vivoand future work will therefore focus on the use of lentiviral delivery systems.

Testicular injection of busulfan for recipient preparation in transplantation of spermatogonial stem cells in mice

2016 ◽  
Vol 28 (12) ◽  
pp. 1916 ◽  
Author(s):  
Yusheng Qin ◽  
Ling Liu ◽  
Yanan He ◽  
Wenzhi Ma ◽  
Huabin Zhu ◽  
...  
Keyword(s):  
Germ Cells ◽  
Fluorescent Protein ◽  
Positive Control ◽  
Negative Control ◽  
Seminiferous Tubules ◽  
Red Fluorescent Protein ◽  
Efferent Duct ◽  
Testicular Injection ◽  
Transplantation Recipient

Intraperitoneal busulfan injections are used to prepare recipients for spermatogonial stem cell (SSC) transplantation but they are associated with haematopoietic toxicity. Testicular injections of busulfan have been proposed to overcome this limitation. To date, testicular injections have not been studied in the mouse model. Therefore, in the present study we used ICR mice as recipients for SSC transplantation and prepared these mice by testicular injection of busulfan on both sides (2, 3, 4 or 6 mg kg–1 per side). Following this, donor germ cells expressing red fluorescent protein (RFP) from transgenic C57BL/6J male mice were transplanted into recipients via the efferent duct on Days 16–17 after busulfan treatment. Positive control mice were prepared by intraperitoneal injection of 40 mg kg–1 busulfan and negative control mice were treated with bilateral testicular injection of 50% dimethyl sulfoxide. On Day 49 after transplantation, recipient mice that were RFP-positive by in vivo imaging were mated with ICR female mice. Donor-derived germ cell colonies with red fluorescence were observed on Day 60 after transplantation, and donor-derived offspring were obtained. The results demonstrated that endogenous germ cells were successfully eliminated in the seminiferous tubules via testicular busulfan administration, and that exogenous SSCs successfully undergo spermatogenesis in the testes of recipient mice prepared by testicular injections of busulfan. In addition to its effects on recipient preparation, this method was safe in rodents and could possibly be adapted for use in other species.

scholarly journals OTX5 Regulates Pineal Expression of the Zebrafish REV-ERBα through a New DNA Binding Site

2008 ◽  
Vol 22 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Shin-ichi Nishio ◽  
Tomoko Kakizawa ◽  
Gilles Chatelain ◽  
Gérard Triqueneaux ◽  
Frédéric Brunet ◽  
...  
Keyword(s):  
Pineal Gland ◽  
Binding Site ◽  
Fluorescent Protein ◽  
Specific Gene ◽  
Orphan Nuclear Receptor ◽  
Cis Acting ◽  
Lower Vertebrates ◽  
Green Fluorescent ◽  
Zebrafish Embryogenesis

Abstract The pineal gland plays a central role in the photoneuroendocrine system and acts as a photosensory organ in lower vertebrates. The orphan nuclear receptor Rev-erbα (NR1D1) has previously been shown to be expressed in the pineal and to be regulated with a robust circadian rhythm during zebrafish embryogenesis. This early pineal expression is under the control of the transcription factor Orthodenticle homeobox 5 (Otx5). In this paper, we show that Otx5 regulates the second zfRev-erbα promoter, ZfP2. Despite the absence of a classical Otx-binding site within ZfP2, this regulation depends on the integrity of the Otx5 homeodomain. Mapping experiments as well as EMSAs show that this interaction between Otx5 and ZfP2 depends on a noncanonical bipartite Otx-binding site (GANNCTTA and TAAA) that we called pineal expression related element (PERE). We showed that PERE is necessary for pineal expression in vivo by injecting zebrafish embryos with wild type and mutated versions of zfRev-erbα promoter fused to green fluorescent protein. Interestingly, PERE is found upstream of other genes expressed in the pineal gland, suggesting that it may play an important role in governing pineal expression. Our data establish that PERE is a novel cis-acting element contributing to pineal-specific gene expression and to Otx target gene regulation.

scholarly journals Effect of Nickel Administration in vivo on the Testicular Structure in Male Mice

2007 ◽  
Vol 76 (2) ◽  
pp. 223-229 ◽  
Author(s):  
P. Massányi ◽  
N. Lukáč ◽  
J. Zemanová ◽  
A. V. Makarevich ◽  
P. Chrenek ◽  
...  
Keyword(s):  
Body Mass ◽  
Seminiferous Tubule ◽  
Basal Membrane ◽  
Relative Volume ◽  
The Body ◽  
Germinal Epithelium ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Group B

The aim of this study was to describe the effects of nickel (NiCl2) on murine testicular structure. Experimental animals were injected intraperitoneally with a single dose of 20 mg NiCl2 per kg of body mass (group A, n = 5) and 40 mg NiCl2 per kg b. m. (group B, n = 5). The group without injection (n = 5) was the control (C). Animals were killed 48 hours after administration of nickel. The body mass of animals, the mass of testes and the testes : body mass ratio were not significantly affected. In both experimental groups a significant (p < 0.001) decrease of germinal epithelium in comparison with control group was observed. The relative volume of the interstitium was increased but not significantly in both experimental groups. An increase in the relative volume of the lumen was registered in both experimental groups in comparison with the control group. The qualitative analysis detected a dilatation of blood vessels in the interstitium, undulation of the basal membrane and several empty spaces in the germinal epithelium. The diameter (n = 150) of the seminiferous tubule was markedly (p < 0.05) decreased in both experimental groups (A, B) compared to control group. The height of the germinal epithelium showed a significant decrease (p < 0.05 - 0.001) after nickel administration. Evaluation of the lumen diameter in the seminiferous tubule showed a significant increase in both experimental groups. The data of the perimeter of seminiferous tubules corresponded with those of the seminiferous tubule diameter. TUNEL assay detected a higher frequency of localized apoptosis in the interstitium of nickel-administered animals compared to control group. Our findings clearly suggest a negative effect of nickel on the structure as well as on the function of the seminferous epithelium at the site of spermatozoa production.

scholarly journals Efficient and Highly Specific Gene Transfer Using Mutated Lentiviral Vectors Redirected with Bispecific Antibodies

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Christina L. Parker ◽  
Timothy M. Jacobs ◽  
Justin T. Huckaby ◽  
Dimple Harit ◽  
Samuel K. Lai
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Endosomal Escape ◽  
Bispecific Antibodies ◽  
Target Cells ◽  
Specific Gene ◽  
Targeted Gene Delivery

ABSTRACT Despite their exceptional potencies, the broad tropism of most commonly used lentivirus (LV) vectors limits their use for targeted gene delivery in vivo. We hypothesized that we could improve the specificity of LV targeting by coupling (i) reduction of their binding to off-target cells with (ii) redirection of the vectors with a bispecific antibody (bsAb) that binds both LV and receptors on target cells. As a proof of concept, we pseudotyped nonreplicating LV using a mutated Sindbis envelope (mSindbis) with ablated binding to native receptors, while retaining the capacity to facilitate efficient fusion and endosomal escape. We then evaluated the transduction potencies of the mSindbis LV for HER2-positive (HER2+) (SKBR3) breast and HER2-negative (HER2−) (A2780) cells when redirected with different bsAbs. mSindbis LV alone failed to induce appreciable green fluorescent protein (GFP) expression in either cell. When mixed with HER2-targeting bsAb, mSindbis LV was exceptionally potent, transducing 12% to 16% of the SKBR3 cells at a multiplicity of infection (MOI [ratio of viral genome copies to target cells]) of 3. Transduction was highly specific, resulting in ∼50-fold-greater selectivity toward SKBR3 cells versus A2780 cells. Redirecting mSindbis LV led to a 10-fold improvement in cell-specific targeting compared to redirecting wild-type Sindbis LV with the same bsAb, underscoring the importance of ablating native virus tropism in order to maximize targeting specificity. The redirection of mutated LV using bsAb represents a potent and highly versatile platform for targeted gene therapy. IMPORTANCE The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentivirus (LV) vectors are efficient gene delivery vehicles but offer little specificity. Here, we report an effective and versatile strategy to redirect LV to target cells using bispecific antibodies (bsAbs) that bind both cell receptors and LV envelope domains. Importantly, we ablated the native receptor binding of LV to minimize off-target transduction. Coupling bsAb specificity and ablated native LV tropism synergistically enhanced the selectivity of our targeted gene delivery system. The modular nature of our bsAb-based redirection enables facile targeting of the same LV to diverse tissues/cells. By abrogating the native broad tropism of LV, our bsAb-LV redirection strategy may enable lentivirus-based gene delivery in vivo, expanding the current use of LV beyond ex vivo applications.

Focal Orchitis in Undescended Testes

2002 ◽  
Vol 126 (1) ◽  
pp. 64-69
Author(s):  
Manuel Nistal ◽  
María Luisa Riestra ◽  
Ricardo Paniagua
Keyword(s):  
Germ Cell ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Seminiferous Tubule ◽  
Immune Cell ◽  
Laboratory Data ◽  
Rete Testis ◽  
Seminiferous Tubules ◽  
Inflammatory Infiltrates ◽  
Lymphoid Infiltrates

Abstract Objective.—To evaluate seminiferous epithelium lesions in adult cryptorchid testes showing lymphoid infiltrates in seminiferous tubules and interstitium (ie, focal orchitis). Also, to consider the possible role of this lesion in the etiology of tubular atrophy. Methods.—We performed a histopathologic study of the cryptorchid testes and adjacent epididymides removed from 50 adult men who had not been previously treated for cryptorchidism. The study included morphologic and semiquantitative evaluation of seminiferous tubule pathology (according to germ cell numbers), Sertoli cell morphology, tubular lumen dilation, rete testis pattern (normal, hypoplastic, or cystic), and epididymal pattern (normal or epididymal duct hypoplasia). The study also included immunohistochemical evaluation of immune cell markers. The results were compared with clinical and laboratory findings. Results.—Focal lymphoid infiltrates (mainly lymphocytes) in seminiferous tubules and interstitium were found in 22 patients (44%), all of whom had unilateral cryptorchidism. The course of orchitis was asymptomatic, and laboratory data were normal. According to the seminiferous tubule pathology, a variety of histopathologic diagnoses, were made: (1) mixed atrophy consisting of Sertoli cell–only tubules intermingled with tubules showing maturation arrest of spermatogonia (11 testes, 4 of which also showed hyalinized tubules); (2) Sertoli cell–only tubules plus hyalinized tubules (4 testes); (3) Sertoli cell–only tubules (3 testes); (4) intratubular germ cell neoplasia (2 testes, 1 of which also showed hyalinized tubules); (5) complete tubular hyalinization (1 testis); and (6) tubular hyalinization plus some groups of tubules with hypospermatogenesis (all germ cell types were present although in lower numbers, 1 testis). Dysgenetic Sertoli cells, that is, Sertoli cells that had undergone anomalous, incomplete maturation, were observed in all nonhyalinized seminiferous tubules with inflammatory infiltrates. Tubular ectasia was observed in 13 cases. The rete testis was hypoplastic and showed cystic transformation in 18 testes, and the epididymis was hypoplastic in 15 testes. Conclusions.—The causes of these focal inflammatory infiltrates are unknown. It is possible that tubular ectasia and Sertoli cell dysgenesis are involved and that these alterations cause a disruption of the blood-testis barrier and allow antigens to enter the testicular interstitium, giving rise to an autoimmune process.

scholarly journals Rat Blood–Brain Barrier Genomics. II

2002 ◽  
Vol 22 (11) ◽  
pp. 1319-1326 ◽  
Author(s):  
Jian Yi Li ◽  
Ruben J. Boado ◽  
William M. Pardridge
Keyword(s):  
Blood Brain Barrier ◽  
Gene Families ◽  
Subtractive Hybridization ◽  
Brain Barrier ◽  
Specific Gene ◽  
Blood Brain ◽  
Tester Cdna ◽  
Junctional Complexes ◽  
Brain Microvasculature

The tissue-specific gene expression at the brain microvasculature, which forms the blood–brain barrier (BBB) in vivo, can be elucidated with a brain vascular genomics program, which starts with the isolation of gene products derived from purified brain microvessels. Genes commonly expressed in peripheral organs are subtracted with the suppression subtractive hybridization method using driver cDNA produced from a pool of rat liver/kidney–derived poly A+RNA. From a screen of 480 clones in the subtracted tester cDNA library, 156 clones were sequenced. The clones fell into 3 groups: known genes (51%), rat expressed sequence tags (31%), and novel rat genes not found in databases (18%). The known genes could be categorized into families of common function including vascular remodeling, signal transduction, transcription factors, biologic transport, vascular amyloid, hemostasis, myelin, lipids, secretion, cytoskeleton, and junctional complexes. Brain vascular genomics, or BBB genomics, allows for an accelerated discovery of the gene families that are differentially expressed at the microvasculature in brain.

Thick ascending limb-specific expression of Cre recombinase

2003 ◽  
Vol 285 (1) ◽  
pp. F33-F39 ◽  
Author(s):  
Peter K. Stricklett ◽  
Deborah Taylor ◽  
Raoul D. Nelson ◽  
Donald E. Kohan
Keyword(s):  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Genomic Library ◽  
Yellow Fluorescent Protein ◽  
Cre Recombinase ◽  
Specific Gene ◽  
Thick Ascending Limb ◽  
Specific Expression ◽  
Enhanced Yellow Fluorescent Protein

Evaluation of thick ascending limb (TAL) function has been hindered by the limited ability to selectively examine the function of this nephron segment in vivo. To address this, a Cre/loxP strategy was employed whereby the Tamm-Horsfall (THP) promoter was used to drive Cre recombinase expression in transgenic mice. The THP gene was cloned from a mouse genomic library, and 3.7 kb of the mouse THP 5′-flanking region containing the first noncoding exon of the THP gene were inserted upstream of an epitope-tagged Cre recombinase (THP-CreTag). THP-CreTag transgenic mice were bred with ROSA26-enhanced yellow fluorescent protein (eYFP) mice (contain a loxP-flanked “STOP” sequence 5′ to eYFP), and doubly heterozygous offspring were analyzed. THP and eYFP were expressed in an identical pattern with predominant localization to the renal outer medulla without expression in nonrenal tissues. eYFP did not colocalize with thiazide-sensitive cotransporter (distal tubule) or neuronal nitric oxide synthase (macula densa) expression. THP mRNA expression was detected only in kidney, whereas CreTag mRNA was also present in testes. These data indicate that THP-CreTag transgenic mice can be used for TAL-specific gene recombination in the kidney.

scholarly journals Characterizing a distal muscle enhancer in the mouse Igf2 locus

2016 ◽  
Vol 48 (2) ◽  
pp. 167-172 ◽  
Author(s):  
Damir Alzhanov ◽  
Peter Rotwein
Keyword(s):  
Skeletal Muscle ◽  
Transcriptional Control ◽  
Fluorescent Protein ◽  
Artificial Chromosome ◽  
Control Element ◽  
Specific Gene ◽  
Reporter System ◽  
Chromosomal Locus ◽  
Igf2 Gene

Insulin-like growth factor-2 (IGF2) is highly expressed in skeletal muscle and was identified as a quantitative trait locus for muscle mass. Yet little is known about mechanisms of its regulation in muscle. Recently, a DNA segment found ∼100 kb from the Igf2 gene was identified as a possible muscle transcriptional control element. Here we have developed an in vivo reporter system to assess this putative enhancer by substituting nuclear (n) EGFP for Igf2 coding exons in a bacterial artificial chromosome containing the mouse Igf2 - H19 chromosomal locus. After stable transfection into a mesenchymal stem cell line, individual clones were converted to myoblasts and underwent progressive muscle-specific gene expression and myotube formation in differentiation medium. Transgenic mRNA and nuclear-targeted enhanced green fluorescent protein were produced coincident with endogenous Igf2 mRNA, but only in lines containing an intact distal conserved DNA element. Our results show that a 294 bp DNA fragment containing two E-boxes is a necessary and sufficient long-range enhancer for induction of Igf2 gene transcription during skeletal muscle differentiation and provides a robust experimental platform for its further functional dissection.

Development of a new micro-injection-system for adenoviral gene delivery (MAGD)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e22167-e22167
Author(s):  
G. Bohlen ◽  
A. Raabe ◽  
J. Dahm-Daphi ◽  
E. Dikomey ◽  
S. E. Schild ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nude Mice ◽  
Fluorescent Protein ◽  
Infusion Pump ◽  
Glass Plate ◽  
Adenoviral Vectors ◽  
Injection System ◽  
Plastic Tube

e22167 Background: Safe and effective delivery of viral vectors is important for gene therapy. Local application of such vectors is more efficient than systemic application. However, currently available injection systems are not optimal because placement of the injection needle and the vector application itself are generally not reproducibile. We developed a new injection system providing controlled injection of adenoviral vectors in tumors (xeno-transplants) in nude mice (NMRI nu/nu). Methods: The new MAGD consists of two fixation devices mounted on a 40x40cm plexi-glass plate, four injection units, and one pump unit with 4 infusion-pumps (Bee hive, Bioanalytical, Chesire, UK). Each injection unit is linked to a pump with commercially available plastic tube. Because the injection arms can be moved in x-,y- and z-direction, virus injection can easily performed for irregular tumor volumes. The entire injection volume is equally distributed to the 4 injection pumps allowing precise and steady injection rates between 0.1μl/min and 100μl/min. Success was confirmed with MRI-scans (Magnetom symphony, Siemens, Erlangen, Germany) 10 min after the injection. The efficacy of the MAGD was tested in 4 human squamous cell carcinoma cell lines from oro-/hypopharynx (FaDu, UD- SCC2, UD-SCC6, and UD-SCC7a). Virus transfection was performed with an adenovirus (serotype 5) expressing enhanced green fluorescent protein (eGFP). Transfection rates were quantified with flow cytometry. Successfully transfected cells expressed eGFP resulting in green fluorescence. In-vitro cell lines (concentration: 10 particles per cell, MOI 10) of FaDu, UD-SCC2, UD-SCC6 and UD-SCC7a served as controls. Results: The MAGD was easy to handle. Injection of 100μl (25μl per infusion pump) at an injection rate of 5μl/min took only 5 min. The in-vivo transfection rates achieved with the MAGD were 9±1% for FaDu, 39±2% for UD- SCC, 54±1% for UD-SCC6 and 54±9% for UD-SCC7a, respectively. The in-vitro transfection rates (controls) for these 4 cell lines were 2±1%, 23±4%, 29±4% and 3±2%, respectively. Conclusions: The new MAGD provided controlled injection of adenoviral vectors in tumors in nude mice. It proved to be effective, as it resulted in comparably high transfection rates. No significant financial relationships to disclose.

scholarly journals Tracking distinct microglia subpopulations with photoconvertible Dendra2 in vivo

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Eric B. Miller ◽  
Sarah J. Karlen ◽  
Kaitryn E. Ronning ◽  
Marie E. Burns
Keyword(s):  
Immune Cells ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Immune Cell ◽  
Neuronal Damage ◽  
Ex Vivo ◽  
Fluorescent Markers ◽  
Immune Cell Subpopulations ◽  
Cell Subpopulations

Abstract Background The ability to track individual immune cells within the central nervous system has revolutionized our understanding of the roles that microglia and monocytes play in synaptic maintenance, plasticity, and neurodegenerative diseases. However, distinguishing between similar subpopulations of mobile immune cells over time during episodes of neuronal death and tissue remodeling has proven to be challenging. Methods We recombineered a photoconvertible fluorescent protein (Dendra2; D2) downstream of the Cx3cr1 promoter commonly used to drive expression of fluorescent markers in microglia and monocytes. Like the popular Cx3cr1–GFP line (Cx3cr1+/GFP), naïve microglia in Cx3cr1–Dendra2 mice (Cx3cr1+/D2) fluoresce green and can be noninvasively imaged in vivo throughout the CNS. In addition, individual D2-expressing cells can be photoconverted, resulting in red fluorescence, and tracked unambiguously within a field of green non-photoconverted cells for several days in vivo. Results Dendra2-expressing retinal microglia were noninvasively photoconverted in both ex vivo and in vivo conditions. Local in vivo D2 photoconversion was sufficiently robust to quantify cell subpopulations by flow cytometry, and the protein was stable enough to survive tissue processing for immunohistochemistry. Simultaneous in vivo fluorescence imaging of Dendra2 and light scattering measurements (Optical Coherence Tomography, OCT) were used to assess responses of individual microglial cells to localized neuronal damage and to identify the infiltration of monocytes from the vasculature in response to large scale neurodegeneration. Conclusions The ability to noninvasively and unambiguously track D2-expressing microglia and monocytes in vivo through space and time makes the Cx3cr1–Dendra2 mouse model a powerful new tool for disentangling the roles of distinct immune cell subpopulations in neuroinflammation.

Sign in / Sign up

Export Citation Format

Share Document