- Research article
- Open Access
Infectivity of adeno-associated virus serotypes in mouse testis
BMC Biotechnology volume 18, Article number: 70 (2018)
Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection.
We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells.
In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.
Adeno-associated viruses (AAVs) are promising gene therapy vectors as AAV-mediated gene delivery is very efficient and safe . However, germ line transmission of the transgene delivered by AAVs is a safety concern in the field [2, 3]. On the other hand, AAVs with limited genome integration  could serve as potent tools to deliver transgenes for generating animal models. Here, transplantation of germ cells engineered in vitro with AAV has been promising [5, 6]. An easier alternative is direct testicular injections of AAVs to target male germ line. In either scenario, gene therapy or transgenesis in animal model, it is crucial to know the infectivity of the AAV serotypes in testis.
AAV are non-enveloped viruses of parvoviridae family with a single-stranded DNA genome of 4.7 kilobases (kb), packaged in a capsid protein of icosahedral symmetry. AAVs possess the ability to infect both dividing and non-dividing cells . Different serotypes of AAVs are known to have tropism towards different cell types . The basis of tropism specificity is the polymorphism of capsid protein. Knowledge of infectivity of different serotypes within a given tissue or organ is valuable in gene therapy context.
The stem cells of the male germ line spermatogonial stem cells (SSCs) and their descendant spermatogonial cells are located within the seminiferous tubules. The tubules are formed by epithelial Sertoli cells. Spermatids produced from spermatogonia reach lumen of the tubules. The tubules are barricaded by an epithelial layer of peritubular contractile myoid cells. The myoid cell epithelia along with the Sertoli cell epithelia form the formidable blood-testis barrier in rodents [9, 10]. Outside the myoid cell barrier, testosterone-producing Leydig cells and blood vessels occupy the interstitial niche among the tubules. Testicular injection introduces the AAVs in the interstitial space exterior to the myoid cell layer. There is scant information on the infectivity of different AAV serotypes in testis.
Here, we report infectivity of a number of AAV serotypes within testis upon injection in mouse testis capsule. Except two, all serotypes tested efficiently target interstitial cells. Specifically, AAV2 and AAV9 uniquely transduced Leydig cells. Notably, a phosphomutant of AAV2 serotype engineered to improve virion survival, displayed a dramatically altered tropism. It traversed myoid cell barrier and infected Sertoli cells, but did not transduce Leydig cells. In spite of direct injection into testis at moderate to high titre, none of the tested serotypes infect SSCs. Thus, our findings support their label as safe vehicles for gene therapy.
Wild type AAVs preferentially target Leydig cells
To investigate the tropism of AAV serotypes in testis and infectivity of sperm progenitors, we injected AAVs of different serotypes into the interstitial space of the mouse testis (Fig. 1a, b; schematics of the experiment, testis cross section). Since, the Sertoli cell mediated blood-testis barrier develops at puberty, we injected 4 weeks old prepubescent animals to test possible viral distribution in the adluminal compartment of seminiferous tubules. Our thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays showed that a large number of sperm progenitors are in the proliferative compartment during this period (Additional file 1: Figure S1A). We tested five different serotypes AAV2, 5, 8, 9 and AAVrh10 at 1 X 109 AAV viral genomes (vgs) per testis (see Methods). All serotypes have enhanced green fluorescent protein (EGFP) expression cassette flanked by AAV2 inverted terminal repeats, but pseudo-typed with capsid proteins of the different serotypes. Majority of the serotypes have been reported to show expression at the site of injection within a week of injection . Therefore, we analyzed bio-distribution of all five serotypes 8 days following injection. Widespread transduction was observed in testes injected with AAV2, 9 and 10 by live GFP expression on whole mount, while AAV5 and 8 showed no or few transduced cells, respectively (Fig. 1c; Additional file 1: Figure S1B). To assess AAV distribution in testis, immunofluorescence was performed on testes cryosections for the virally encoded GFP. For AAV2, 9 and 10, our analysis revealed GFP+ transduced cells uniquely in the intertubular spaces after 8 days (Fig. 1d; Additional file 1: Figure S1B). To quantitate the transduction efficiency we enumerated GFP+ cells on cryosections. In accordance with the wholemount GFP expression, AAV2, 9 and 10 showed higher efficiency compared to AAV 5 and 8 (Additional file 1: Figure S1C; see Fig. 2c for AAV2). Next, we addressed the specific cell type transduced in the testis. Staining with lipophilic Nile red showed that testosterone-producing Leydig cells, which contain large lipid droplets are targeted by AAV2 and AAV9 (Fig. 1d). However, endothelial cells of the vasculature immunostained with CD31, also in the intertubular space, are not targeted by AAV2 or AAV9 (Fig. 1e). Thus, it appears that the unique target population of AAVs, at least of AAV2 and AAV9 serotypes, are Leydig cells outside the seminiferous tubules and that they do not infect tubules or intratubular cells.
The myoid cell layer around the tubules is a potential barrier that could shield tubules from AAVs. In fact, the peritubular myoid cells circumscribe the developing seminiferous tubules and begin to express Collagen 1 in foetal testis [11, 12]. While, Collagen 1 component of the extracellular matrix (ECM) derives exclusively from myoid cells, Laminin is secreted by Sertoli cells . We have performed AAV injections at 4 weeks. By this stage, the myoid cell epithelial barrier and ECM are fully formed as shown by comparison of 3, 4 and 8 weeks old testes using smooth muscle actin-α (myoid cell layer), Collagen 1 and Laminin 5 (ECM markers) as well as integrin β1 (apical membrane of Sertoli cells; Additional file 2: Figure S2). Thus, it is likely that AAVs fail to traverse the peritubular myoid cell or the ECM barrier.
To test the impact of increasing the viral titer on distribution within testis, we injected AAV2 at 1 X 1010 AAV viral genomes (vgs) per testis and analyzed 8 days post injection. At this higher titer, AAV2 showed similar transduction pattern of GFP+ cells uniquely in the interstitium (Fig. 2a, b). Sal4+ SSCs outlining the periphery of the tubules clearly showed that increasing the titer did not result in viral infiltration across myoid cell barrier nor transduction of SSCs (Fig. 2b). However, quantitation of GFP+ cells revealed more efficient transduction with higher titer (Fig. 2c). Higher titer AAV5 injection (1010 vgs per testis) resulted in rare GFP+ cells in interstitial space (Fig. 2d). Thus, most wildtype AAV serotypes tested infect intertubular cell types and did not infiltrate across the myoid layer.
Engineered AAV2 mutant displays novel tropism in testis
The viral particles are targeted for ubiquitin-mediated proteasome degradation machinery by phosphorylation of specific residues on the capsid. Mutations of these phosphodegrons improve the transduction efficiency of AAV2 as well as other serotypes [14,15,16]. In parallel to testing wildtype serotypes, we had tested one mutant each of AAV2 and AAV9 for infectivity in testis. AAV2 TM is a triple mutant with residue changes S489A, T251A and K532R, while AAV9 mutant was an S499A modification described previously [17, 18]. The mutants used here, showed improved transduction on cultured HeLa cells or in hepatic gene transfer in mice over their wildtype counterparts [17, unpublished data]. The mutant serotypes were injected in the testes of 4 weeks old males and analyzed 1 month later. AAV9 S499A mutant is less efficient in transducing cells in murine testis compared to its cognate wildtype serotype (Fig. 3a, c; Additional file 1: Figure S1C). Remarkably, we found GFP+ cells in the tubules upon testicular injection of AAV2 TM (Fig. 3a, b). Moreover, Nile red staining showed that AAV2 TM did not infect Leydig cells (Fig. 4a). To determine the cell type infected by AAV2 TM within the seminiferous tubules, cryosections were stained with a membrane marker, wheat germ agglutinin (WGA) that binds to N-acetyl-D-glucosamine on plasma membrane and has been extensively used to identify spermatogenic cells . Confocal imaging showed GFP expression in the epithelial Sertoli cells with the typical branched morphology, extending from the basement membrane to the lumen of the tubules (Fig. 4b). However, no GFP expression was observed in sperm progenitors at different stages of differentiation, which are brightly marked by WGA (Fig. 4b). Thus, we conclude that AAV2 TM primarily target Sertoli cells and not SSCs or its derivatives. Overall, the results indicate a fully altered tropism for AAV2 within testis upon mutation of the residues chosen to improve virion infectivity.
Evaluating the infectivity of AAV serotypes in various organs and tissues is key to exploit this nucleic acid delivery vehicle for gene therapies. Assessing preferential tropism of AAVs in testis is key to asses safety of AAVs from the perspective of vertical gene transmission. In contrast, serotypes with a germ line targeting potential will be valuable for achieving stable transgenesis in animal models using CRISPR (clustered regularly interspaced short palindromic repeats) technology. Our data attest to the safety of AAVs as gene therapy vehicles. However, we show striking alteration in tropism in testis upon engineering AAV capsids for improving transduction. Thus, our report emphasizes the need for reassessment of tropism of engineered AAVs to ensure they are safe for gene therapies.
Our data represents a thorough analysis of distribution of a set of AAVs when injected directly in the testis capsule. This mode of delivery allows more rigorous evaluation of infectivity within testis than systemic delivery. We show that AAV2 and AAV9 efficiently transduce testosterone-producing Leydig cells. Endothelia of blood vessels occupying the same interstitial space as Leydig cells are not transduced revealing specificity of AAV2 and AAV9 for the testosterone producing cells. None of the tested wildtype AAVs appeared to infiltrate into seminiferous tubules or infect sperm progenitors. It is possible that the CAG chimeric promoter-enhancer sequence used to drive GFP reporter is not expressed in the sperm progenitors. However, a recent work has shown that this enhancer is active in sperm progenitors . Remarkably, this study shows transduction of SSCs and downstream sperm progenitors by AAV1 and AAV9 . We speculate that the higher titres used by this study compared to that in our experiments may explain the apparent conflict in the results between the two studies. Moreover, the dilution of episomal rAAV vector genomes in proliferating sperm progenitors may have further reduced the possibility of detecting AAV9 transduction of SSCs in our study.
Differential tropism of AAV serotypes arises due to capsid protein polymorphism . We show that specific mutations of putative phosphodegrons on AAV2 capsid confers novel infectivity. Notably, the triple mutant AAV2 crosses myoid barrier and infects Sertoli cells but does not transduce Leydig cells revealing a complete switch in tropism within testis compared to its cognate wildtype. Thus, our observations suggest that it is possible to engineer AAVs to target SSCs for transgenesis in animal models. In fact, rational design based on knowledge of ligand-receptor interactions mediating virus entry, as well as high-throughput screening using capsid peptide-display combined with selection by directed evolution, both have been successful approaches to engineer AAVs of desired tropism [21, 22]. On the other hand, the finding that mutations aimed to increase the transduction by averting proteasome-mediated degradation significantly changes infectivity of a specific AAV serotype underscores the importance of screening altered tropism while designing AAVs with increased stability or increased transduction efficiency.
Although AAV2 serotype is considered a safe gene therapy vector with respect to vertical transmission, it is documented to transduce murine spermatognia-derived cells in culture . This infectivity may be due to the removal of myoid cell barrier and / or due to the culture-induced changes in spermatogonia-derived cells, because, our study clearly shows that wild type AAV2 does not transduce them in vivo. In rodents, myoid cells form a single layer with tight junctions and this epithelium-like layer is a component of the blood-testis barrier [9, 10]. Therefore, it is likely that many wildtype AAVs do not efficiently cross the peritubular myoid barrier and thus, attest to their safety in the context of gene therapy.
The alteration in tropism in a capsid mutant designed to improve transduction highlights the necessity of rigorous assessment of tropism of engineered AAVs. Importantly, our results provide a strong support for existing literature and affirm safety of AAV gene therapy vectors owing to its low germ line transmission potential. Nevertheless, we have not assayed for the presence of rAAV genomic DNA in germ cells. This is important for future studies aimed at assessing the risk of germ line transmission of individual serotypes.
Animals were sourced from the mouse facility at the Institute of Stem Cell Biology and Regenerative Medicine (InStem). F1 hybrids of C57BL/6 J and DBA2J were used for experiments. These inbred strains were originally sourced from The Jackson Laboratory, USA and subsequently, maintained as inbred colonies at the InStem facility. Animals were euthanized by CO2 inhalation as per the institutional guidelines.
The AAV helper plasmids were from Agilent technologies (Stratagene, Santa Clara, CA, USA) and the AAV packaging plasmids were a kind gift from Dr. Arun Srivastava, University of Florida, Gainesville. Highly purified stocks of self-complementary wild-type (WT) AAVs or the mutant AAV vectors encoding the enhanced green fluorescent protein (EGFP) gene driven by the chicken β-actin promoter containing the CMV enhancer and SV40 poly A signal were generated by polyethyleneimine based triple transfection of AAV-293 cells (Stratagene). Briefly, 40 dishes (150mm2) of 80% confluent AAV 293 cells were transfected with AAV rep-cap plasmid, transgene containing plasmid and AAV-helper free (p.helper) plasmid. Cells were collected 72 h post transfection, lysed and treated with 25 units/ml of benzonase nuclease (Sigma Aldrich, St Louis, MO, USA). Subsequently, the vectors were purified by iodixanol gradient ultra-centrifugation  (Optiprep, Sigma Aldrich) followed by column chromatography (HiTrap Q column, GE Healthcare, Pittsburgh, PA). The vectors were finally concentrated to a final volume of 0.5 ml in phosphate buffered saline (PBS) using Amicon Ultra 10 K centrifugal filters (Millipore, Bedford, MA). The physical particle titres of the vectors were quantified independently thrice by slot blot analysis and the mean value was expressed as viral genomes (vgs)/ml . Site-directed mutagenesis was performed to generate AAV2 Triple mutant vector containing the S489A, T251A and K532R mutations and a single mutant AAV9 S499A [17, 18] using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s protocol. Briefly, a one-step PCR amplification of the target sites was performed for 18 cycles with the primers (sequence available on request) followed by DpnI digestion for 1 h. 2 μL of this digested PCR product was then transformed into XL10-Gold Ultracompetent Cells (Stratagene). Following plasmid isolation, the presence of the desired point mutation was verified by DNA sequencing (Applied Biosystems 3130 Genetic Analyzer, Warrington, UK).
Male mice, F1 hybrids of C57BL/6 J and DBA2J were used for experiments and injections performed as previously reported . The animals were anesthetized by Isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane), surgical site was sterilized with ethanol and topical application of Betadine. A single incision was made on the ventral skin and body wall about 1.5 cm anterior to the genitals, using sterile surgical scissors under aseptic conditions. The testes were pulled from the scrotal sac holding the fat pad. The volumes of the viral stocks were adjusted with PBS to achieve either 1 X 109 vgs or 1 X 1010 in 15 μl volume. Each testis was injected 15 μl of the viral suspension using 30G needle syringe. The typical titre we obtain in AAV preparations in laboratory scale is 1011–1012 viral genomes / ml and the upper limit of the injection volume in the mouse testis capsule is 15 μl. Injection was into intertubular spaces, also known as testis capsule. In a set of animals, left testis served as an uninjected control. The animals were sacrificed after desired period of incubation (3 or 8 days or 4 weeks) post injection and testes were dissected for analysis.
Immunostaining and fluorescence microscopy
Testes were dissected and imaged wholemount for GFP expression using Stereo zoom microscope Leica M205FA and Leica DFC 3000G monochrome camera. For immunostaining analysis, testes were fixed in 4% PFA in PBS for 45 min at 4 °C and thoroughly washed with PBS. The samples were embedded in Tissue-Tek O.C.T. compound and snap cryofrozen. Cryosections of 10–16 μm thickness were taken on Superfrost plus slides and analysed by fluorescence immunostaining. Random transverse sections were chosen for analysis. For PLZF antibody staining, antigen retrieval was done, wherein the sections were boiled in a solution of 10 mM sodium citrate, pH 6.5 for 3 min. For all staining, blocking and permeabilization was performed in PBS containing 10% FBS and 0.5% TritonX100. Then, sections were immunostained overnight with primary antibody. Antibodies used were: chick- GFP (Abcam; ab13970, 1/1000), PLZF (PG Pelicci, IFOM, Italy, 1/700), Collagen I (Abcam; ab21286, 1/250), smooth muscle actin-α (Abcam; ab137734, 1/200), integrin β1 (Abcam; ab95623, 1/150). The sections were then washed and incubated with secondary antibodies (Donkey raised Alexa-Fluor antibodies from Molecular probes, 1/500). For Nile red staining (Sigma; 7248), following secondary antibody sections were incubated with 0.5 μg/ml Nile red in PBS for 10 min at room temperature. For wheat germ agglutinin staining (WGA), sections were incubated with WGA in PBS for 1 h, the tissues were post-fixed with 4% paraformaldehyde for 15 min and then, incubated with primary and secondary antibody. EdU was injected intraperitoneally at a concentration of 200 μg / g body weight of animals (Injection volume 100 μl). Four EdU pulses were administered in 48 h (12 h interval), animals were sacrificed and the dissected testes were cryosectioned for analysis. EdU incorporation was analysed using Click-iT® EdU Alexa Fluor® 488 Imaging Kit as per the manufacturer’s instruction (ThermoFisher Scientific) following immunostaining. The slides were mounted in 75% Tris-buffered glycerol and images were acquired using Olympus FV100 confocal microscope and Olympus IX73 or BX53 fitted with Olympus DP26 or DP72 camera using CellSens software.
Quantitation and statistical analysis
For each testis, the number of GFP+ cells per 10X field (0.58 mm2) was averaged across 3 fields. Mean value from n = 3 animals is represented with standard deviation. Statistical analysis was performed using GraphPad Prism 5.0 Software. Unpaired t test with Welch’s correction was performed at 5% level of significance (p < 0.05) to assess significance of observed difference in the infectivity between moderate and high titre serotypes.
(enhanced) green fluorescent protein
Extra cellular matrix
Spermatogonial stem cell
Wheat germ agglutinin
Clustered regularly interspersed short palindromic repeats
Hastie E, Samulski RJ. AAV at 50: a golden anniversary of discovery, research, and gene therapy success, a personal perspective. Hum Gene Ther. 2015;26(5):257–65.
Arruda VR, Fields PA, Milner R, Wainwright L, De Miguel MP, Donovan PJ, et al. Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males. Mol. Ther. 2001;4(6):586–92.
Jakob M, Muhle C, Park J, Weiss S, Waddington S, Schneider H. No evidence for germ-line transmission following prenatal and early postnatal AAV-mediated gene delivery. J Gene Med. 2005;7(5):630–7.
Schultz BR, Chamberlain JS. Recombinant adeno-associated virus transduction and integration. Mol Ther. 2008;16(7):1189–99.
Adeno-associated virus-mediated delivery of genes to mouse spermatogonial stem cells. Biol. Reprod. 2017;96(1):221–31.
Honaramooz A, Megee S, Zeng W, Destrempes MM, Overton SA, Luo J, et al. Adeno-associated virus (AAV)-mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation. FASEB J. 2008;22(2):374–82.
Zinn E, Vandenberghe LH. Adeno-associated virus: fit to serve. Curr Opin Virol. 2014;8:90–7.
Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther. 2008;16(6):1073–80.
Dym M, Fawcett DW. The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol Reprod. 1970;3(3):308–26.
Tindall D, Rowley D, Murthy L, Lipshultz L, Chang C. Structure and biochemistry of the Sertoli cell. Int Rev Cytol. 1985;94:127–49.
Jeanes A, Wilhelm D, Wilson MJ, Bowles J, McClive PJ, Sinclair AH, et al. Evaluation of candidate markers for the peritubular myoid cell lineage in the developing mouse testis. Reproduction. 2005;130(4):509–16.
Martineau J, Nordqvist K, Tilmann C, Lovell-Badge R, Capel B. Male-specific cell migration into the developing gonad. Curr Biol. 1997;7(12):958–68.
Skinner MK, Tung PS, Fritz IB. Cooperativity between Sertoli cells and testicularperitubular cells in the production and deposition ofextracellular matrix components. J Cell Biol. 1985;100(6):1941–7.
Sen D, Balakrishnan B, Gabriel N, Agrawal P, Roshini V, Samuel R, et al. Improved adeno-associated virus (AAV) serotype 1 and 5 vectors for gene therapy. Sci Rep. 2013;3:1832.
Sen D, Gadkari RA, Sudha G, Gabriel N, Kumar YS, Selot R, et al. Targeted modifications in adeno-associated virus serotype 8 capsid improves its hepatic gene transfer efficiency in vivo. Hum. Gene Ther. Methods. 2013;24(2):104–16.
Gabriel N, Hareendran S, Sen D, Gadkari RA, Sudha G, Selot R, et al. Bioengineering of AAV2 capsid at specific serine, threonine, or lysine residues improves its transduction efficiency in vitro and in vivo. Hum. Gene Ther. Methods. 2013;24:80–93.
Balakrishnan B, Krishnagopal A, Sen D, Srivastava A, Jayandharan GR. Improved AAV1 and AAV9 vectors for gene delivery. Am. Soc. Gene Cell Therpy. 2013. p. Mol Ther 21 Suppl S130–131.
Sen D, Kalaivani V, Gadkari RA, Sudha G, Srinivasan N, Srivastava A, et al. Multiple molecular alterations in phosphodegrons 1–3 within AAV2 capsid demonstrates higher hepatic gene transfer efficiency. Am. Soc. Gene Cell Therpy. 2013. p. Mol Ther 21 suppl S206–207.
Lee M-C, Damjanov I. Anatomic distribution of lectin-binding sites in mouse testis and epididymis. Differentiation. 1984;27:74–81.
Watanabe S, Kanatsu-Shinohara M, Ogonuki N, Matoba S, Ogura A, Shinohara T. In vivo genetic manipulation of Spermatogonial stem cells and their microenvironment by adeno-associated viruses. Stem Cell Reports. 2018;10(5):1551–64.
Einfeld DA, Schroeder R, Roelvink PW, Lizonova A, King CR, Kovesdi I, et al. Reducing the native tropism of adenovirus vectors requires removal of both CAR and integrin interactions. J Virol. 2001;75(23):11284–91.
Liu Y, Siriwon N, Rohrs JA, Wang P. Generation of targeted adeno-associated virus (AAV) vectors for human gene therapy. Curr Pharm Des. 2015;21(22):3248–56.
Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chesnut K, et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 1999;6(6):973–85.
Kube DM, Srivastava A. Quantitative DNA slot blot analysis: inhibition of DNA binding to membranes by magnesium ions. Nucleic Acids Res. 1997;25(16):3375–6.
Sehgal L, Thorat R, Khapare N, Mukhopadhaya A, Sawant M, Dalal SN. Lentiviral mediated transgenesis by in vivo manipulation of spermatogonial stem cells. PLoS One. 2011;6(7):e21975.
We thank NCBS / inStem Animal Care and Resource Center; Central Imaging and Flow Cytometry Facility at NCBS; Sorab Dalal and Rahul Thorat, ACTREC, Mumbai for initiating us on testicular injections; Drs. Srikala Raghavan, Mitradas Panicker and Colin Jamora for antibodies and dyes. We thank G. Kalaivani, Dept. of Haematology, Christian Medical College, Vellore for generation of AAV vectors.
JT, JP, SSR and MJ were funded by inStem. RS is a Ramalingswami fellow, Department of Biotechnology (DBT; http://www.dbtindia.nic.in/), India (BT/RLF/Re-entry/03/2010). The work was supported by inStem (www.instem.res.in); Animal work was partially supported by the National Mouse Research Resource (NaMoR) grant (BT/PR5981/MED/31/181/2012;2013–2016) from the DBT. The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
All experiments were performed in accordance with institutional safety guidelines and with the approval of Institutional Biosafety Committee of Institute of Stem Cell Biology and Regenerative Medicine (InStem). Animal work: All procedures performed in this study were approved by the InStem Institutional Animal Ethics Committee (IAEC) following norms specified by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The reference for the IAEC approval accorded to the study is inStem/RK-2/2014.
Consent for publication
The authors have no competing interests to declare.
Figure S1. Distribution of transduction efficiency of AAVs in mouse testis. A) Immunostaining with EdU staining. Edu incorporation and staining in 4 to 5 weeks old males reveals proliferating sperm progenitors in the seminiferous tubules outlined by Laminin 5 staining. B) Top: Wholemount of dissected testis imaged for live GFP. Bottom: Cryosection immunostained for GFP. Scale bars 100 μm. C) Histogram representing the transduction efficiency of the various serotypes. For each testis, the number of GFP+ cells in a 10X field was enumerated; the number GFP+ cells / 0.58 mm2 (mean ± standard deviation; AAV5, 0; AAV8, 26 ± 14.1; AAV9, 104 ± 24.1; AAV9 S499A, 45.3 ± 10.4; AAVrh10, 78 ± 15.1; n = 3 animals; n = 2 animals for AAV 8). (PDF 2291 kb)
Figure S2. Immunostained cross sections of testis at 3-, 4- and 8- week-old adult testis. α-Smooth muscle actin (α-Sma) is a myoid cell marker. Collagen 1 and Laminin 5 mark the ECM and β1 integrin marks the periphery of seminiferous tubules. Scale bar 50 μm. For 3 right panels of the 4 week time point, scale bar 100 μm. (PDF 6157 kb)
About this article
Cite this article
Rajasekaran, S., Thatte, J., Periasamy, J. et al. Infectivity of adeno-associated virus serotypes in mouse testis. BMC Biotechnol 18, 70 (2018). https://doi.org/10.1186/s12896-018-0479-1
- Adeno-associated viruses (AAV)
- Germline transmission
- Mouse testis injection
- Myoid cell
- Leydig cell
- Sertoli cell