Light controls mesophyll-specific post-transcriptional splicing of photoregulatory genes by AtPRMT5.

Light plays a central role in plant growth and development, providing an energy source and governing various aspects of plant morphology. Previous study showed that many polyadenylated full-length RNA molecules within the nucleus contain unspliced introns (post-transcriptionally spliced introns, PTS introns), which may play a role in rapidly responding to changes in environmental signals. However, the mechanism underlying post-transcriptional regulation during initial light exposure of young, etiolated seedlings remains elusive. In this study, we used FLEP-seq2, a Nanopore-based sequencing technique, to analyze nuclear RNAs in Arabidopsis (Arabidopsis thaliana) seedlings under different light conditions and found numerous light-responsive PTS introns. We also used single-nucleus RNA sequencing (snRNA-seq) to profile transcripts in single nucleus and investigate the distribution of light-responsive PTS introns across distinct cell types. We established that light-induced PTS introns are predominant in mesophyll cells during seedling de-etiolation following exposure of etiolated seedlings to light. We further demonstrated the involvement of the splicing-related factor A. thaliana PROTEIN ARGININE METHYLTRANSFERASE 5 (AtPRMT5), working in concert with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a critical repressor of light signaling pathways. We showed that these two proteins orchestrate light-induced PTS events in mesophyll cells and facilitate chloroplast development, photosynthesis, and morphogenesis in response to ever-changing light conditions. These findings provide crucial insights into the intricate mechanisms underlying plant acclimation to light at the cell-type level.

CSFV restricts necroptosis to sustain infection by inducing autophagy/mitophagy-targeted degradation of RIPK3.

IMPORTANCE: CSFV infection in pigs causes persistent high fever, hemorrhagic necrotizing multi-organ inflammation, and high mortality, which seriously threatens the global swine industry. Cell death is an essential immune response of the host against pathogen invasion, and lymphopenia is the most typical clinical feature in the acute phase of CSFV infection, which affects the initial host antiviral immunity. As an "old" virus, CSFV has evolved mechanisms to evade host immune response after a long genetic evolution. Here, we show that necroptosis is a limiting host factor for CSFV infection and that CSFV-induced autophagy can subvert this host defense mechanism to promote its sustained replication. Our findings reveal a complex link between necroptosis and autophagy in the process of cell death, provide evidence supporting the important role for CSFV in counteracting host cell necrosis, and enrich our knowledge of pathogens that may subvert and evade this host defense.

Role of OGDH in Atophagy-IRF3-IFN-ß pathway during classical swine fever virus infection.

Classical swine fever (CSF) is a severe infectious disease caused by the classical swine fever virus (CSFV) that poses significant challenges to the swine industry. α-ketoglutarate dehydrogenase (OGDH), the first rate-limiting enzyme of the tricarboxylic acid (TCA) cycle, catalyzes α-ketoglutarate (α-KG) to succinyl-CoA, playing a crucial role in glycometabolism. Our previous studies showed that CSFV disrupts the TCA cycle, resulting in α-KG accumulation. However, the interplay between CSFV and OGDH remains unclear. In this study, we found that CSFV significantly reduces OGDH protein levels and promotes α-KG secretion through OGDH in PK-15 cells. Furthermore, we observed CSFV C protein interacts with OGDH and revealed that CSFV utilizes NDP52/NBR1 to target OGDH protein degradation in the autophagy-lysosome pathway. We also unveiled that OGDH overexpression inhibits CSFV proliferation, whereas OGDH knockdown increases CSFV proliferation. Further investigation into the mechanisms of OGDH on CSFV replication revealed that OGDH regulates the AMPK-mTOR-autophagy pathway. Additionally, using the autophagy agonist/inhibitor, rapamycin/3-MA, we observed that OGDH modulates autophagy to regulate the IRF3-IFN-ß network and CSFV replication. These findings shed light on the role of OGDH in CSFV infection and host metabolism, promoting the development of innovative strategies for combating CSFV and other viral infections via targeting metabolic pathways.

Arable soil nitrogen dynamics reflect organic inputs via the extended composite phenotype.

Achieving food security requires resilient agricultural systems with improved nutrient-use efficiency, optimized water and nutrient storage in soils, and reduced gaseous emissions. Success relies on understanding coupled nitrogen and carbon metabolism in soils, their associated influences on soil structure and the processes controlling nitrogen transformations at scales relevant to microbial activity. Here we show that the influence of organic matter on arable soil nitrogen transformations can be decoded by integrating metagenomic data with soil structural parameters. Our approach provides a mechanistic explanation of why organic matter is effective in reducing nitrous oxide losses while supporting system resilience. The relationship between organic carbon, soil-connected porosity and flow rates at scales relevant to microbes suggests that important increases in nutrient-use efficiency could be achieved at lower organic carbon stocks than currently envisaged.

Genome-wide characterization of nascent RNA processing in plants.

Following transcription initiation, RNA polymerase II (Pol II) elongates through the genic region and terminates after the polyadenylation signal. This process is accompanied by splicing, 3' cleavage, and polyadenylation, to eventually form a mature mRNA. Recent advances in short-read and long-read high-throughput sequencing methods have shed light on the global landscape of these co-transcriptional events at nucleotide resolution. In this mini review, we summarize recent developments in genome-wide approaches that broadened our understanding of nascent RNA processing in plants.

The Advances of Broad-Spectrum and Hot Anti-Coronavirus Drugs.

Coronaviruses, mainly including severe acute respiratory syndrome virus, severe acute respiratory syndrome coronavirus 2, Middle East respiratory syndrome virus, human coronavirus OC43, chicken infectious bronchitis virus, porcine infectious gastroenteritis virus, porcine epidemic diarrhea virus, and murine hepatitis virus, can cause severe diseases in humans and livestock. The severe acute respiratory syndrome coronavirus 2 is infecting millions of human beings with high morbidity and mortality worldwide, and the multiplicity of swine epidemic diarrhea coronavirus in swine suggests that coronaviruses seriously jeopardize the safety of public health and that therapeutic intervention is urgently needed. Currently, the most effective methods of prevention and control for coronaviruses are vaccine immunization and pharmacotherapy. However, the emergence of mutated viruses reduces the effectiveness of vaccines. In addition, vaccine developments often lag behind, making it difficult to put them into use early in the outbreak. Therefore, it is meaningful to screen safe, cheap, and broad-spectrum antiviral agents for coronaviruses. This review systematically summarizes the mechanisms and state of anti-human and porcine coronavirus drugs, in order to provide theoretical support for the development of anti-coronavirus drugs and other antivirals.

Viral Vector Vaccine Development and Application during the COVID-19 Pandemic.

With the accumulation of mutations in SARS-CoV-2 and the continuous emergence of new variants, the importance of developing safer and effective vaccines has become more prominent in combating the COVID-19 pandemic. Both traditional and genetically engineered vaccines have contributed to the prevention and control of the pandemic. However, in recent years, the trend of vaccination research has gradually transitioned from traditional to genetically engineered vaccines, with the development of viral vector vaccines attracting increasing attention. Viral vector vaccines have several unique advantages compared to other vaccine platforms. The spread of Omicron has also made the development of intranasal viral vector vaccines more urgent, as the infection site of Omicron is more prominent in the upper respiratory tract. Therefore, the present review focuses on the development of viral vector vaccines and their application during the COVID-19 pandemic.

Accelerating the pace of ecotoxicological assessment using artificial intelligence.

Species Sensitivity Distribution (SSD) is a key metric for understanding the potential ecotoxicological impacts of chemicals. However, SSDs have been developed to estimate for only handful of chemicals due to the scarcity of experimental toxicity data. Here we present a novel approach to expand the chemical coverage of SSDs using Artificial Neural Network (ANN). We collected over 2000 experimental toxicity data in Lethal Concentration 50 (LC50) for 8 aquatic species and trained an ANN model for each of the 8 aquatic species based on molecular structure. The R2 values of resulting ANN models range from 0.54 to 0.75 (median R2 = 0.69). We applied the predicted LC50 values to fit SSD curves using bootstrapping method, generating SSDs for 8424 chemicals in the ToX21 database. The dataset is expected to serve as a screening-level reference SSD database for understanding potential ecotoxicological impacts of chemicals.

Development of Recombinase Aided Amplification Combined With Disposable Nucleic Acid Test Strip for Rapid Detection of Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is the dominant causative agent of PCV2 systemic disease (PCV2-SD) in pigs. It can also associate with other diseases such as respiratory and enteric diseases, reproductive failure, porcine dermatitis and nephropathy syndrome in pigs. Currently, PCV2 infection is a considerable threat in the swine industry. Therefore, it is of great significance to prevent, control, and accurately detect PCV2 in pig farms. Recombinase aided amplification (RAA) technology is an isothermal nucleic acid amplification technology that could rapidly amplify the target gene fragment at a constant temperature. The amplification products labeled with specific molecules could be visually detected using the test strip with the corresponding antibody. In the present study, the RAA technology combined with a nucleic acid test strip (RAA-strip) was established for simple and specific detection of PCV2. Primers and probes targeting the PCV2 ORF2 gene were designed according to the RAA technology principles. The PCV2 RAA-strip established in this study could detect as low as 103 copies/µL of recombinant plasmids containing the PCV2 ORF2 gene fragment. The lowest detection limit about viral DNA and virus titers was 6.7 × 10-6 ng/µL and 10 TCID50/mL, respectively. Furthermore, no cross-reaction with other porcine viruses occurred at 37°C and within 15 min. We used 42 clinical samples to assess the performance of our established method. The positive rate of clinical samples detected by PCV2 RAA-strip was 50.00%. This was similar to that detected by PCV2 PCR (45.24%). In conclusion, due to the advantages of strong specificity, high sensitivity, excellent reproducibility, and simple operation method, our PCV2 RAA-strip is suitable for the rapid clinical detection of PCV2 on-site.

Development and Application of RAA Nucleic Acid Test Strip Assay and Double RAA Gel Electrophoresis Detection Methods for ASFV and CSFV.

African swine fever (ASF) is an acute, severe and hemorrhagic infectious disease caused by African swine fever virus (ASFV) infecting domestic pigs and wild boars. Since the outbreak of the disease in China in 2018, it has brought a great impact on China's pig industry. Classical swine fever (CSF) is an acute contact infectious disease of pigs caused by classical swine fever virus (CSFV) infection. Clinically, acute CSF usually shows persistent high fever, anorexia, extensive congestion and bleeding of the skin and mucosa, which are similar to ASF. It is of great significance to prevent, control and accurately detect ASF and CSF in pig farms. In this study, Recombinase aided amplification (RAA) technology combined with a nucleic acid test strip (RAA-strip) was established for simple and specific detection of ASFV/CSFV. The sensitivity and preliminary clinical application results showed that the RAA test strip established in this study could detect recombinant plasmids containing ASFV/CSFV gene fragments as low as 103 copies/µL. The minimum detection limits of virus DNA/cDNA were 10 and 12 pg respectively, and there was no cross-reaction with other porcine viruses. The specificity of the method was good. We used 37-42 clinical samples to evaluate the performance of our established method, and the positive concordance rates with conventional PCR were 94.1 and 57.1%, respectively. In addition, ASFV and CSFV double RAA agarose gel electrophoresis detection methods were established. The results showed that the method had good specificity. The detection limit of this method is 106 copies for ASFV p72 gene recombinant plasmid and 105 copies for CSFV NS5B Gene recombinant plasmid. The use of this method for clinical material detection was consistent with the PCR method. In summary, the developed method of RAA-strip assay for ASFV and CSFV realized the visual detection of pathogens, and the developed method of dual RAA agarose gel electrophoresis assay for ASFV and CSFV realized the simultaneous detection of two pathogens in one reaction, with good specificity, high sensitivity and rapid reaction rate, which was expected to be clinically feasible for the differential diagnosis of ASF and CSF provided technical support.

Classical swine fever virus employs the PERK- and IRE1-dependent autophagy for viral replication in cultured cells.

Endoplasmic reticulum stress (ERS)-mediated autophagy is indispensable for modulation of replication and pathogenesis of numerous mammalian viruses. We have previously shown that classical swine fever virus (CSFV) infection induces ERS-mediated autophagy for maintaining viral replication both in vivo and in vitro, however, the underlying mechanism remains unclarified. Here we found that CSFV infection activates the PERK pathway-dependent complete autophagy to promote viral replication in cultured PK-15 and 3D4/2 cells. Likewise, our results also suggested the essential roles of the IRE1/GRP78-mediated complete autophagy in CSFV replication in vitro. Furthermore, we suggested that CSFV infection induces activation of the PERK and IRE1 pathway for potential immunoregulation via promoting transcription of proinflammatory cytokine (IFN-γ and TNF-α) genes in the CSFV-infected cells. Finally, pharmacological treatment of PERK- or IRE1-pathway regulators, and the corresponding SiRNAs interventions did not affect the viabilities of the cells, excluding the potential interference elicited by altered cell viabilities. Taken together, our results suggest that CSFV infection induces complete autophagy through activation of the PERK and IRE1 pathway to facilitate viral replication in cultured cells, and modulation of proinflammatory cytokines may be a potential mechanism involved in this event. Our findings will open new horizons for molecular mechanisms of sustainable replication and pathogenesis of CSFV, and lay a theoretical foundation for the development of ERS-autophagy-targeting therapeutic strategies for clinical control of CSF.

Melatonin protects the mouse testis against heat-induced damage.

Spermatogenesis, an intricate process occurring in the testis, is responsible for ongoing production of spermatozoa and thus the cornerstone of lifelong male fertility. In the testis, spermatogenesis occurs optimally at a temperature 2-4°C lower than that of the core body. Increased scrotal temperature generates testicular heat stress and later causes testicular atrophy and spermatogenic arrest, resulting in a lower sperm yield and therefore impaired male fertility. Melatonin (N-acetyl-5-methoxytryptamine), a small neuro-hormone synthesized and secreted by the pineal gland and the testis, is widely known as a potent free-radical scavenger; it has been reported that melatonin protects the testis against inflammation and reactive oxygen species generation thereby playing anti-inflammatory, -oxidative and -apoptotic roles in the testis. Nevertheless, the role of melatonin in the testicular response to heat stress has not been studied. Here, by employing a mouse model of testicular hyperthermia, we systematically investigated the testicular response to heat stress as well as the occurrence of autophagy, apoptosis and oxidative stress in the testis. Importantly, we found that pre-treatment with melatonin attenuated heat-induced apoptosis and oxidative stress in the testis. Also, post-treatment with melatonin promoted recovery of the testes from heat-induced damage, probably by maintaining the integrity of the Sertoli cell tight-junction. Thus, we for the first time provide the proof of concept that melatonin can protect the testis against heat-induced damage, supporting the potential future use of melatonin as a therapeutic drug in men for sub/infertility incurred by various testicular hyperthermia factors.

Classical Swine Fever Virus Infection Induces Endoplasmic Reticulum Stress-Mediated Autophagy to Sustain Viral Replication in vivo and in vitro.

Endoplasmic reticulum (ER) stress-mediated autophagy plays significant roles in replication and pathogenesis of many animal viruses. However, the relationship between ER stress, autophagy, and viral replication during in vivo and in vitro infection of classical swine fever virus (CSFV) remains unclear. In this study, we established a pig model for CSFV infection and found that viral loads of CSFV differed in 10 kinds of infected organs, and that the degree of tissue lesions was to some extent positively correlated with CSFV replication in vivo. Next, we found that CSFV infection not only induced ER stress and subsequently activated three unfolded protein responses (UPR) pathways including protein kinase R-like ER kinase (PERK), inositol requiring enzyme 1 (IRE1), and activating transcription factor-6 (ATF-6) pathways, but also triggered complete autophagy in main immune organs and partial nonimmune organs exhibiting severer pathological injuries and higher viral loads. However, only the IRE1 pathway and no autophagy were activated in some other nonimmune organs with slighter pathologies and lower viral loads. These results indicate a potential link between CSFV-induced ER stress and autophagy, both of which are associated with the CSFV replication in vivo. We further performed in vitro experiments and found that CSFV infection activates the PERK and IRE1 pathways and autophagy in cultured porcine kidney cell lines (PK-15) and macrophage cell lines (3D4/2), and pharmacological regulation of ER stress remarkably changed autophagic activities induced by CSFV, suggesting that CSFV-induced autophagy can be mediated by ER stress possibly via the PERK and IRE1 pathway. Furthermore, treatment with ER stress regulators significantly altered copy numbers of NS5B genes, expression of Npro proteins, and viral titers in CSFV-infected cells or in cells treated with autophagy regulators prior to CSFV infection, suggesting the requirement of ER stress-mediated autophagy for CSFV replication in vitro. Collectively, our data demonstrate that CSFV induces ER stress-mediated autophagy to sustain its replication in vivo and in vitro, which may be one of the potential strategies exploited by CSFV for immune evasion. This finding will provide new insights into mechanisms of replication and pathogenesis of CSFV, and development of new strategies for controlling CSF.

Does the use of pre-calculated uncertainty values change the conclusions of comparative life cycle assessments? - An empirical analysis.

In life cycle assessment (LCA), performing Monte Carlo simulation (MCS) using fully dependent sampling typically involves repeated inversion of a technology matrix for a sufficiently large number of times. As the dimension of technology matrices for life cycle inventory (LCI) databases grows, MCS using fully dependent sampling is becoming a computational challenge. In our previous work, we pre-calculated the distribution functions of the entire LCI flows in the ecoinvent ver. 3.1 database to help reduce the computation time of running fully dependent sampling by individual LCA practitioners. However, it remains as a question whether the additional errors due to the use of pre-calculated uncertainty values are large enough to alter the conclusion of a comparative study, and, if so, what is the odds of such cases. In this study, we empirically tested the probability of altering the conclusion of a comparative LCA due to the use of pre-calculated uncertainty values. We sampled 10,000 random pairs of elementary flows of ecoinvent LCIs (ai and bi) and ran MCSs (1) using pre-calculated uncertainty values and (2) using fully dependent sampling. We analyzed the distribution of the differences between ai and bi (i.e., ai-bi) of each run, and quantified the probability of reversing (e.g., ai > bi became ai < bi) or moderating the conclusion (e.g., ai > bi became ai ≈ bi). In order to better replicate the situation under a comparative LCA setting, we also sampled 10,000 random pairs of elementary flows from the processes that produce electricity, and repeated the same procedure. The results show that no LCIs derived using pre-calculated uncertainty values constitute large enough differences from those using fully dependent sampling to reverse the conclusion. However, in 5.3% of the cases, the conclusion from one approach is moderated under the other approach or vice versa. When elementary flow pairs are sampled only from the electricity-producing processes, the probability of moderating the conclusions increases to 10.5%, while that of reversing the conclusions remains nil. As the number of unit processes in LCI databases increases, running full MCSs in a PC-environment will continue to be a challenge, which may lead some LCA practitioners to avoid uncertainty analysis altogether. Our results indicate that pre-calculated distributions for LCIs can be used as a proxy for comparative LCA studies in the absence of adequate computational resources for full MCS. Depending on the goal and scope of the study, LCA practitioners should consider using pre-calculated distributions if the benefits of doing so outweighs the associated risks of altering the conclusion.

Electrically tunable polarizer based on graphene-loaded plasmonic cross antenna.

The unique gate-voltage dependent optical properties of graphene make it a promising electrically-tunable plasmonic material. In this work, we proposed in situ control of the polarization of nanoantennas by combining plasmonic structures with an electrostatically tunable graphene monolayer. The tunable polarizer is designed based on an asymmetric cross nanoantenna comprising two orthogonal metallic dipoles sharing the same feed gap. Graphene monolayer is deposited on a Si/SiO2 substrate, and inserted beneath the nanoantenna. Our modelling demonstrates that as the chemical potential is incremented up to 1 eV by electrostatic doping, resonant wavelength for the longer graphene-loaded dipole is blue shifted for 500 nm (~10% of the resonance) in the mid-infrared range, whereas the shorter dipole experiences much smaller influences due to the unique wavelength-dependent optical properties of graphene. In this way, the relative field amplitude and phase between the two dipole nanoantennas are electrically adjusted, and the polarization state of the reflected wave can be electrically tuned from the circular into near-linear states with the axial ratio changing over 8 dB. Our study thus confirms the strong light-graphene interaction with metallic nanostructures, and illuminates promises for high-speed electrically controllable optoelectronic devices.

Phospholipase D Family Member 6 Is a Surface Marker for Enrichment of Undifferentiated Spermatogonia in Prepubertal Boars.

Spermatogonial stem cells (SSCs) have a functional ability to maintain self-renewal and sustain production of spermatozoa throughout the reproductive lifespan of a male. Studies on SSCs can thus better the understandings of spermatogenesis and unravel the mechanisms for self-renewal and differentiation of male germline stem cells. However, the rarity of SSCs in the testis and the lack of reliable surface markers obstruct the related study and further application of SSCs. This is especially the case in livestock animals. In this study, we identified that phospholipase D family member 6 (PLD6) is a surface marker for undifferentiated spermatogonia in boar testes. By magnetic-activated cell sorting, PLD6+ cell fraction comprises 84.45% ± 0.35% of undifferentiated spermatogonia (marked by PLZF). Xenotransplantation of PLD6+ cells into the recipient mouse testis revealed a ninefold increase of donor cell-derived colony formation compared with that in the unselected cell group, indicating the significant enrichment of SSCs. Furthermore, based on the sorted PLD6+ cells with a high SSC content, we established a feeder-free culture system that could maintain porcine undifferentiated spermatogonia for 4 weeks in vitro with the expression of typical markers throughout the culture period. In conclusion, this study demonstrates that PLD6 is a surface marker of undifferentiated spermatogonia in testes of prepubertal boars and could be utilized to unprecedentedly enrich porcine undifferentiated spermatogonia. These data provide the basis for future studies on the refinement of germ cell culture and manipulation of porcine undifferentiated spermatogonia.

Dynamic Model for the Stocks and Release Flows of Engineered Nanomaterials.

Most existing life-cycle release models for engineered nanomaterials (ENM) are static, ignoring the dynamics of stock and flows of ENMs. Our model, nanoRelease, estimates the annual releases of ENMs from manufacturing, use, and disposal of a product explicitly taking stock and flow dynamics into account. Given the variabilities in key parameters (e.g., service life of products and annual release rate during use) nanoRelease is designed as a stochastic model. We apply nanoRelease to three ENMs (TiO2, SiO2 and FeOx) used in paints and coatings through seven product applications, including construction and building, household and furniture, and automotive for the period from 2000 to 2020 using production volume and market projection information. We also consider model uncertainties using Monte Carlo simulation. Compared with 2016, the total annual releases of ENMs in 2020 will increase by 34-40%, and the stock will increase by 28-34%. The fraction of the end-of-life release among total release flows will increase from 11% in 2002 to 43% in 2020. As compared to static models, our dynamic model predicts about an order of magnitude lower values for the amount of ENM released from this sector in the near-term while stock continues to build up in the system.

Long-Term Propagation of Porcine Undifferentiated Spermatogonia.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis and fertility throughout the adult life of a male. Genetic manipulations of SSCs combined with germ cell transplantation present a novel approach for gene therapy and production of genetically modified animals. However, the rarity of SSCs within mammalian testes remains an impediment to related applications, making in vitro expansion of SSCs a prerequisite. Nevertheless, long-term culture systems of SSCs from large animals have not been established yet. In this study, we developed an optimized in vitro culture condition for porcine undifferentiated spermatogonia. The germ cells were isolated and enriched from 7-day-old porcine testes by an optimized differential planting. We tested different feeder layers and found that neonatal autologous Sertoli cells acted better than the SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cell line and adult Sertoli cells. The effects of several growth factors were also investigated. Using neonatal Sertoli cells as feeder and Dulbecco's modified eagle medium: nutrient mixture F-12 (DMEM/F12) culture medium supplemented with 10% KSR and four cytokines, the undifferentiated spermatogonia can proliferate in vitro for at least 2 months without loss of stemness. The expression of SSC markers indicated that the cultured cells maintained SSC expression profiles. Moreover, xenotransplantation and in vitro induction showed that the long-term cultured cells preserved the capacity to colonize in vivo and differentiate in vitro, respectively, demonstrating the presence of SSCs in the cultured cells. In conclusion, the conditions described in this study can support the normal proliferation of porcine undifferentiated spermatogonia with stemness and normal karyotype for at least 2 months. This culture system will serve as a basic refinement in the future studies and facilitate studies on SSC biology and genetic manipulation of male germ cells.

An efficient strategy for generation of transgenic mice by lentiviral transduction of male germline stem cells in vivo.

BACKGROUND: Male germline stem cells (MGSCs) are a subpopulation of germ cells in the testis tissue. MGSCs are capable of differentiation into spermatozoa and thus are perfect targets for genomic manipulation to generate transgenic animals. METHOD: The present study was to optimize a protocol of production of transgenic mice through transduction of MGSCs in vivo using lentiviral-based vectors. The recombinant lentiviral vectors with either EF-1 or CMV promoter to drive the expression of enhanced green fluorescent protein (eGFP) transgene were injected into seminiferous tubules or inter-tubular space of 7-day-old and 28-day-old mouse testes. At 5 or 6 wk post-surgery, these pre-founders were mated with wild-type C57BL/6J female mice (1.5 to 2.0-month-old). RESULTS: Sixty-seven percent of F1 generation and 55.56 % of F2 offspring were positive for eGFP transgene under the control of EF-1 promoter via PCR analysis. The transgenic pups were generated in an injection site-and age-independent manner. The expression of transgene was displayed in the progeny derived from lentiviral vector containing CMV promoter to drive transgene, but it was silenced or undetectable in the offspring derived from lentiviral vector with transgene under EF-1 promoter. The methylation level of gDNA in the promoter region of transgene was much higher in the samples derived lentiviral vectors with EF-1 promoter than that with CMV promoter, suggesting eGFP transgene was suppressed by DNA methylation in vivo. CONCLUSION: This research reported here an effective strategy for generation of transgenic mice through transduction of MGSCs in vivo using lentivirus vectors with specific promoters, and the transgenic offspring were obtained in an injection site-and age-independent manner. This protocol could be applied to other animal species, leading to advancement of animal transgenesis in agricultural and biomedical fields.