Biology subjects

Zang, X.

Publications and source records attributed to Zang, X..

16 recordsLinked to original sources

bioRxiv2026

Biosynthetic additive manufacturing through mycelium-bacteria consortia

Lignocellulosic biomass is the most abundant renewable raw material on Earth, yet its structural and carbon-storage potential remains unrealized: microbial decomposition returns stored carbon to the atmosphere before it can be harnessed as a durable material. Distinct microbial processes, such as biosynthesis and biomineralization, offer a natural counterpoint, converting organic carbon into mechanically robust, persistent materials such as polymers and minerals. Here, we establish a biosynthetic additive manufacturing strategy through mycelium-bacteria consortia, by coupling fungal self-assembly with bacterial biomineralization inside fabricated three-dimensional scaffolds derived from lignocellulosic biomass. Trametes versicolor forms a continuous living mycelial network that serves as both a structural template and a transport pathway, guiding the spatial colonization of Sporosarcina pasteurii and enabling volumetric calcium carbonate deposition throughout the scaffold interior, overcoming the surface-limited mineralization of conventional microbial systems. Integration with helicoidal Bouligand architectures translates microscale mineral reinforcement into macroscale performance, increasing compressive Young's modulus by 7-14-fold and ultimate strength by 5-fold relative to unmineralized conventional mycelium-based living composites. The living mycelial network also enables autonomous self-healing and modular self-assembly through hyphal bridging without external adhesives or energy input. This work establishes a scalable route for converting and upcycling transient biomass carbon into durable living structural materials for sustainable construction.

bioengineering↗

bioRxiv2026

An accumbal cholinergic ISR-HCN2 axis sustains maladaptive cue motivation

Environmental cues associated with rewarding outcomes can exert persistent control over behavior long after the reward is absent, yet the cellular adaptations that stabilize such maladaptive motivation remain unclear. Here we identify nucleus accumbens cholinergic interneurons (CINs) as causal regulators of persistent cue-driven seeking. Self-administration of addictive drugs or high-fat reward, but not sucrose, selectively increased CIN pacemaker firing. Cocaine-paired cues evoked rapid acetylcholine release in the nucleus accumbens core, and bidirectional manipulation of CINs suppressed or enhanced cue-induced cocaine seeking. Mechanistically, cocaine experience engaged the integrated stress response (ISR) in CINs, increased HCN2 expression and Ih, and stabilized a hyperexcitable state. CIN-specific ISR suppression or HCN2 knockdown reduced cocaine seeking, whereas HCN2 overexpression enhanced it. Local HCN blockade and systemic treatment with the clinically approved HCN blocker ivabradine reduced cue-driven seeking across addictive-drug and high-fat-reward paradigms while sparing sucrose seeking. These findings identify an ISR-HCN2-dependent cholinergic mechanism that stabilizes maladaptive cue-driven motivation and nominate HCN-dependent excitability as a tractable target for therapeutic intervention.

neuroscience↗

bioRxiv2026

Experimental Time Points Guided Transcriptomic Velocity Inference

Time-series single-cell RNA sequencing enables longitudinal tracking of biological processes, yet cellular trajectory reconstruction informed by experimental time remains challenging. Existing trajectory inference methods either perform de novo reconstruction without leveraging experimental time points, or prioritize transitions between time points while paying less attention to intra-time-point dynamics. To reconcile experimental time points with local precision, we present CellDyc, a semi-supervised learning framework that leverages experimental time-point supervision to reconstruct transcriptomic velocities and recover an intrinsic gene-embedded time. CellDyc consistently outperforms existing approaches in reconstructing cellular trajectories across development, disease, and reprogramming contexts. Biologically, CellDyc provides novel insights, such as resolving temporal heterogeneity in erythroid maturation and quantitatively demonstrating that the immunosuppressive environment delays monocyte differentiation in glioblastoma. CellDyc integrates seamlessly with downstream tools like CellRank and remains robust even when only inferred temporal information is available. Collectively, CellDyc offers a rigorous, data-driven solution for deciphering time-resolved cellular dynamics.

bioinformatics↗

bioRxiv2025

TIGIT expression dictates the immunosuppressive reprogramming of myeloid cells in glioblastoma

Glioblastoma (GBM) is a deadly brain cancer with near-universal recurrence despite maximal treatment for which new innovations are sorely needed. Immunotherapy has yet to make significant gains in GBM treatment despite revolutionizing other cancer therapies, due in part to GBM-mediated immune suppression. This immune derangement proceeds through several mechanisms, but increasing evidence points to critical roles for tumor-derived extracellular vesicles (EVs) and immunosuppressive myeloid cells as key factors in this process. In the present study, we demonstrate broad expression of TIGIT across myeloid cell populations in the GBM microenvironment, a finding recapitulated by conditioning healthy monocytes with GBM-derived EVs. Further, knockdown of TIGIT expression reduced the immunosuppressive polarization of monocytes, resulting in improvement in T cell function. This finding proceeded in an NLRP3-dependent manner, with substantial co-localization of TIGIT and NLRP3 expression prior to knockdown. These findings point to a novel role for TIGIT expression in diverse myeloid cells in the GBM microenvironment as a marker of immunosuppressive activity and further indicate a hierarchy of immunomodulatory protein activity in these myeloid cells, with TIGIT knockdown unmasking the pro-inflammatory activity of NLRP3. This study bolsters understanding of the immunosuppressive complexities of myeloid cells in the GBM microenvironment, while lending further support to prevention or attenuation of immunosuppressive myeloid cell activity as a means of restoring immune function in GBM. Graphical abstract(Created in BioRender. Asad, M. (2025) https://BioRender.com/euiljoq O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/648993v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@13dc389org.highwire.dtl.DTLVardef@1f06765org.highwire.dtl.DTLVardef@14200forg.highwire.dtl.DTLVardef@144f245_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LITumor-mediated immune suppression is a key barrier to the development of effective immunotherapies for GBM. C_LIO_LITIGIT is broadly expressed in myeloid cells within the GBM microenvironment and can be induced by GBM-derived extracellular vesicles. C_LIO_LIKnockdown of TIGIT reduces immunosuppressive polarization of monocytes and enhances T cell activity via NLRP3 signaling, implicating TIGIT expression as a targetable modifier of immunosuppressive activity in GBM-associated myeloid cells. C_LI Importance of the StudyGlioblastoma (GBM) remains a formidable clinical challenge, with poor prognosis and limited response to current immunotherapies. This study uncovers a novel immunosuppressive axis involving TIGIT expression in myeloid cells, which are key players in the GBM tumor microenvironment. By demonstrating GBM-derived extracellular vesicles induce TIGIT in healthy monocytes and TIGIT knockdown diminishes immunosuppressive polarization in an NLRP3-dependent manner, this study highlights TIGIT as both a marker and modulator of immune dysfunction in GBM. These findings introduce a functional hierarchy of immunoregulatory proteins in tumor-associated myeloid cells, positioning TIGIT as a potential checkpoint target. Restoring immune activity by disrupting this axis could enhance the efficacy of immunotherapy in GBM. Thus, this research not only advances our understanding of tumor-induced immune suppression but also opens a promising therapeutic avenue to reinvigorate anti-tumor immunity in a cancer type historically resistant to immunotherapeutic approaches.

cancer biology↗

bioRxiv2025

Biodegradable Poly(β-amino amide)s Enable Efficient RNA Delivery and Spleen Targeting

RNA therapeutics are emerging as transformative modalities in clinical applications and have become a key area in life science research. While lipid nanoparticles (LNPs) have become the leading platform for RNA delivery in preventive and therapeutic products, they still face significant challenges in achieving efficient extrahepatic delivery and maintaining long term shelf stability. Here we report the development of a series of biodegradable poly({beta}-amino amide) (PBAA) polymers, detailing their design, synthesis, and performance as gene delivery vehicles both in vitro and in vivo. These cationic polymers, featuring interspersed trialkylamine motifs, provide a readily tunable functional handle and facilitate complexation with gene cargo. The redox-sensitive disulfide motifs introduce a redox-responsive decomposition pathway for the polymer backbone, triggering cargo release during intracellular delivery. The results herein demonstrate that these polymers offer remarkable efficiency in encapsulating and delivering translation-competent cargos, including mRNA and CRISPR-Cas based gene editing tools. Notably, a dodecyl modified PBAA transporter has achieved over 97% gene editing efficiency in vitro, and over 97% spleen-targeting selectivity in a murine model. Additionally, it produces stable nanoparticles that maintain their physicochemical properties at 4 {degrees}C for up to two weeks without addition of excipients such as PEG, offering a cost-effective solution for RNA therapeutics supply chains. As the transformative impact of RNA and other nucleic acids continues to build within the pharmaceutical industry and beyond, it is imperative that the supporting technologies evolve in stride to maximize said impact. The tunable and biodegradable PBAA polymer designs presented herein are illustrative examples of how high-level functional performance can be acheived in conjunction with the critical targeting, formulation, and operational simplicity needed of state-of-the-art transfection and delivery technologies.

biochemistry↗

bioRxiv2024

Efficient mRNA Delivery In Vitro and In Vivo Using a Polycharged Biodegradable Nanomaterial

As RNA rises as one of the most significant modalities for clinical applications and life science research, efficient tools for delivering and integrating RNA molecules into biological systems become essential. We hereby report a formulation using a polycharged biodegradable nano-carrier, N1-501, which demonstrates superior efficiency and versatility in mRNA encapsulation and delivery in both cell and animal models. N1-501 is a polymeric material designed to function through a one-step facile formulation process suitable for various research settings. Its capability for mRNA transfection is investigated across a wide range of mRNA doses and in different biological models, including 18 tested cell lines and mouse models. This study also comprehensively analyzes N1-501s application for mRNA transfection by examining factors such as buffer composition and pH, incubation condition, and media type. Additionally, N1-501s superior in vivo mRNA transfection capability ensures its potential as an efficient and consistent tool for advancing mRNA-based therapies and genetic research.

biochemistry↗

bioRxiv2024

Biosynthetic characterization and combinatorial biocatalysis of the cysteine protease inhibitor E-64

E-64 is an irreversible and selective cysteine protease inhibitor prominently used in chemical biology and drug discovery. In this work, we uncovered and characterized the NRPS-independent pathway responsible for biosynthesis of E-64, which is widely conserved in fungi. Heterologous reconstitution and biochemical assays show the pathway starts with epoxidation of fumaric acid to the warhead (2S,3S)-trans-epoxysuccinic acid with an -ketoglutarate (KG)/Fe(II)-dependent oxygenase, followed by successive condensation with an O_SCPLOWLC_SCPLOW-amino acid by an ATP-grasp enzyme, and with an amine by the first characterized amide bond synthetase from fungi. Both amide bond-forming enzymes displayed significant biocatalytic potential, including scalability, stereoselectivity towards the warhead and broader substrate scopes in forming the amide bonds. Combinatorial biocatalysis with the two amide-bond forming enzymes generated a library of cysteine protease inhibitors and led to more potent analogs towards cathepsin B. In addition, preparative synthesis of clinically relevant cysteine protease inhibitors was accomplished from a single reaction mixture. Our work highlights the importance of biosynthetic investigation for enzyme discovery and the potential of amide bond-forming enzymes as biocatalysts for a library synthesis of small molecules.

biochemistry↗

bioRxiv2022

Study on endogenous inhibitors against PD-L1: cAMP as a potential candidate

The discovery of new anticancer drugs targeting the PD-1/PD-L1 pathway has been research hotspots. In this study, a combination of biological affinity ultrafiltration (BAU), UPLC-HRMS, molecular dynamic (MD) simulations and molecular docking methods were applied to search for endogenous active compounds that can inhibit the binding of PD-L1 and PD-1. We screened dozens of potential cancer related endogenous compounds. The results showed that cyclic adenosine monophosphate (cAMP) had a direct inhibition effect on the PD-1/PD-L1 binding with an in vitro IC50 value of about 2.7 {micro}M determined by homogeneous time-resolved fluorescence (HTRF) assay. The binding mode analyses for the cAMP - dimeric/monomeric PD-L1 complex indicated that cAMP was likely to bind to the dimeric PD-L1, since the binding free energies of the cAMP - dimeric and monomeric PD-L1 complex were about 23.6 and 15.1 kcal/mol, respectively, from MD simulations. The direct binding assay using surface plasmon resonance (SPR) method showed that cAMP could also bind to monomeric PD-L1 fixed on the sensor chip surface with a KD value of about 1.72 mM. Our findings suggested that cAMP may directly inhibit the PD-1/PD-L1 interaction.

molecular biology↗

bioRxiv2022

Functional Contribution of Cancer-Associated Fibroblasts in Glioblastoma

The abundance and biological contribution of cancer associated fibroblasts (CAFs) in glioblastoma are poorly understood. Here, we applied single-cell RNA sequencing and spatial transcriptomics analyses to identify and characterize CAFs in human glioblastoma tumors and then performed functional enrichment analysis and in vitro assays to investigate their interactions with malignant glioblastoma cells. We found that CAF abundance was significantly correlated with tumor grade, poor clinical outcome, and activation of extracellular matrix remodeling, using three large databases containing bulk RNA-sequencing data and clinical information. Proteomic analysis of the CAFs and their secretome revealed fibronectin (FN1) as a strong candidate mediating CAF functions. This was validated using in vitro cellular models, which demonstrated that CAF conditioned media and recombinant FN1 could facilitate the migration and invasion of glioblastoma cells. In addition, we showed that CAFs were more abundant in the mesenchymal-like state (or subtype) than in other states of glioblastomas, while cell lines resembling the proneural-state responded to the CAF signaling better in terms of the migratory and invasive phenotypes. Investigating the in-situ expression of gene markers specifically associated with CAFs and mesenchymal malignant cells further indicated that CAFs were enriched in the perinecrotic and pseudopalisading zones of human tumors, where mesenchymal-like glioblastoma cells co-resided and thus likely interacted. Overall, this study characterized the molecular features and functional impacts of CAFs in glioblastoma, alluding to a novel cell-to-cell interaction axis mediated by CAFs in the glioblastoma microenvironment.

cancer biology↗

bioRxiv2022

Duration reproduction under memory pressure: Modeling the roles of visual memory load in duration encoding and reproduction

Duration estimates are often biased by the sampled statistical context, yielding the classical central-tendency effect, i.e., short durations are over- and long duration underestimated. Most studies of the central-tendency bias have primarily focused on the integration of the sensory measure and the prior information, without considering any cognitive limits. Here, we investigated the impact of cognitive (visual working-memory) load on duration estimation in the duration encoding and reproduction stages. In four experiments, observers had to perform a dual, attention-sharing task: reproducing a given duration (primary) and memorizing a variable set of color patches (secondary). We found an increase in memory load (i.e., set size) during the duration-encoding stage to increase the central-tendency bias, while shortening the reproduced duration in general; in contrast, increasing the load during the reproduction stage prolonged the reproduced duration, without influencing the central tendency. By integrating an attentional-sharing account into a hierarchical Bayesian model, we were able to predict both the general over- and underestimation and the central-tendency effects observed in all four experiments. The model suggests that memory pressure during the encoding stage increases the sensory noise, which elevates the central-tendency effect. In contrast, memory pressure during the reproduction stage only influences the monitoring of elapsed time, leading to a general duration over-reproduction without impacting the central tendency.

neuroscience↗

bioRxiv2021

Reovirus sensitizes microsatellite stable colorectal cancer to anti-PD-1 treatment via cross-talk in innate and adaptive immune systems

BackgroundMicrosatellite stable (MSS) colorectal cancer (CRC) represents ~85% of all CRCs. These tumors are poorly immunogenic and largely resistant to immunotherapy, necessitating a need to develop new immune enhancing strategies. Oncolytic reovirus has a high propensity to replicate in KRAS mutant tumors which account for ~50% of MSS CRCs. Current study explores the ability of reovirus to potentiate the effect of immune checkpoint inhibition in MSS CRC. MethodsEffectiveness of reovirus infection was quantified through MTT assay for cell viability, and expression of immune-response genes by flow cytometry, RT-qPCR, and microarray. Computational analysis of differentially expressed genes was performed by TAC, DAVID and STRING. Combinatorial approach using anti-PD-1 monoclonal antibody was assessed in ex vivo and in vivo models. Live-cell imaging, tumor volume and survival were measured for quantification of anti-tumor activity. Expression of pattern recognition receptors (PRRs), cell surface and activation markers of immune cells, and PD-1/PD-L1 axis were studied using multi-color flow cytometry, immunoblotting, immunohistochemistry, and immunofluorescence. ResultsReovirus infection exerted growth arrest and expression of immune-response genes in CRCs cell lines in a KRAS-dependent manner. However, microsatellite instability, rather than KRAS status determined immune-repose pathways, functionalities and biological processes post-reovirus infection. Furthermore, reovirus significantly enhanced the anti-tumor activity of anti-human PD-1 [nivolumab] treatment in MSS CRC cell lines ex vivo. Similarly, reovirus increased the activity of anti-mouse PD-1 treatment in the CT26 [MSS, KRASMut], but not the MC38 [MSI, KRASWt] syngeneic mouse model of CRC. Combinatorial treatment has reduced the proliferative index, increased apoptosis and differentially altered PD-L1/PD-1 signaling among CT26 and MC38 tumors. Activation of innate immune system and expression of PRRs and antigen presentation markers were observed under reovirus and anti-PD-1 treatment that additionally reduced immunosuppressive macrophages. This led to an increase in T cell subsets, increase in effector T cell activation, and decrease in exhaustion markers specifically within CT26 microenvironment. ConclusionThe current study systematically evaluates immune characteristics and immune microenvironment of CRC under reovirus/anti-PD-1 combination treatment that proves increased effectiveness among MSS compared to MSI CRCs. This is a promising regimen warranting translation into clinical trials. One Sentence SummaryOncolytic reovirus alters innate and adaptive immune system and potentiates MSS type colorectal cancer to checkpoint inhibition therapy.

cancer biology↗

bioRxiv2021

CRISPRa screen on a genetic risk locus shared by multiple autoimmune diseases identifies a dysfunctional enhancer that affects IRF8 expression through cooperative lncRNA and DNA methylation machinery

Dysregulated transcription factors represent a major class of drug targets that mediate the abnormal expression of many critical genes involved in SLE and other autoimmune diseases. Although strong evidence suggests that natural human genetic variation affects basal and inducible gene expression, it is still a considerable challenge to establish a biological link between GWAS-identified non-coding genetic risk variants and their regulated gene targets. Here, we combine genetic data, epigenomic data, and CRISPR activation (CRISPRa) assays to screen for functional variants regulating IRF8 expression. Using CRISPR-mediated deletion and 3D chromatin structure analysis, we demonstrate that the locus containing rs2280381 is a cell-type-specific distal enhancer for IRF8 that spatially interacts with the IRF8 promoter. Further, rs2280381 mediates IRF8 expression through enhancer RNA AC092723.1, which recruits TET1 to the IRF8 promoter to modulate IRF8 expression by affecting methylation levels. The alleles of rs2280381 modulate PU.1 binding and chromatin state to differentially regulate AC092723.1 and IRF8 expression. Our work illustrates a strategy to define the functional genetic variants modulating transcription factor gene expression levels and identifies the biologic mechanism by which autoimmune diseases risk genetic variants contribute to the pathogenesis of disease.

genetics↗

bioRxiv2021

Elevated concentration and nitrogen addition affects responses of foliar phosphorus fractions in invasive species to increased phosphorus supply

No studies have explored how the invasive species of Mikania micranatha and Chromolaena odoratan adjust leaf phosphorus (P) among inorganic P (Pi) and organic P fractions to adapt the low soil P availability, especially under elevated CO2 concentrations ([CO2]) and nitrogen (N) deposition. Here, we address this by measuring foliar total N and P concentrations as well as functional P fractions (i.e. Pi, metabolic P, lipid P, nucleic acids P, and residual P) of both invasive species and a native species (Paederia. scandens) growing under different P supplies, N, and N+P addition under both ambient and elevated [CO2]. Phosphorus addition greatly increased plant biomass and foliar P concentrations but did not significantly affect foliar N concentration and leaf mass per unit leaf area (LMA). In response to P addition, the concentration of metabolic P increased the most, followed by that of nucleic acid P, Pi, and lipid P, in all species by an average of 754%, 82%, 53%, and 38%, respectively. However, elevated [CO2] and N addition weakened this positive effect on concentrations of foliar P fractions in the invasive species. Our results indicate that elevated [CO2] and N addition allowed the invasive species to acclimate to a low soil P availability, supporting their successful invasion, through greatly reducing P allocation to non-metabolic foliar P fractions (phospholipids and nucleic acid P) to meet their demand for metabolic P and Pi for photosynthesis, rather than altering LMA.

plant biology↗

bioRxiv2019

Effect of recombinant phycocyanin on photosynthetic system in Chlamydomonas reinhardtii

The phycobilisome is an important photosynthetic antenna in the photosynthetic cyanobacteria, and phycocyanin is one of the main components of phycobilisomes. It helps cells absorb green light that green-lineage photo-synthetic organisms cannot. In this work, phycocyanin, heme oxidase and ferredoxin oxidoreductase from Arthrospira platensis FACHB 314 were successfully expressed in the green algae Chlamydomonas reinhardtii. Then the effects of this expression on the photosynthesis and growth of C. reinhardtii were detected. Transcriptional level analysis showed that the phycocyanin gene was successfully expressed stably in the transgenic strains. The results of low-temperature fluorescence emission spectra and chlorophyll fluorescence showed that recombinant phycocyanin has considerable optical activity. The expression of phycocyanin, heme oxidase and ferredoxin oxidoreductase in low-light conditions is particularly evident in the promotion of photosynthesis in C. reinhardtii. The growth of transgenic strains was significantly promoted in the early growth phase under low-light conditions. However, the final growth and biomass accumulation of transgenic C. reinhardti were inhibited by this expression. In this paper, the possibility of photoenergy transfer between phycocyanin and heterologous host thylakoid membrane was researched, which provided a useful attempt for the construction of a new photosynthetic system using phycobiliprotein from cyanobacteria.\n\nOne-sentence summaryPhycocyanin from Arthrospira platensis FACHB 314 expressed in Chlamydomonas reinhardtii can effect the photosynthetic system of C. reinhardtii.

molecular biology↗

bioRxiv2019

Translational regulation of non-autonomous mitochondrial stress response promotes longevity

Inhibition of mRNA translation delays aging, but the underlying mechanisms remain underexplored. Mutations in both DAF-2 (IGF-1 receptor) and RSKS-1 (ribosomal S6 kinase/S6K) cause synergistic lifespan extension in C. elegans. To understand the roles of S6K-mediated translational regulation in this process, we performed genome-wide translational profiling and genetic screens to identify genes that are not only regulated at the translational level in the daf-2 rsks-1 mutant, but also affect lifespan. Inhibition of CYC-2.1 (cytochrome c) in the germline significantly extends lifespan through non-autonomous activation of the mitochondrial unfolded protein response (UPRmt) and AMP-activated kinase (AMPK) in the metabolic tissue. Furthermore, the RNA-binding protein GLD-1-mediated translational repression of cytochrome c in the germline is important for the non-autonomous activation of UPRmt and synergistic longevity of the daf-2 rsks-1 mutant. Together, these results illustrate a translationally regulated non-autonomous mitochondrial stress response mechanism in the modulation of lifespan by insulin-like signaling and S6K. HighlightsO_LILongevity of the daf-2 rsks-1 mutant is mediated by translational repression of ribosomal proteins and CYC-2.1/cytochrome c. C_LIO_LIGermline inhibition of cyc-2.1 non-autonomously activates UPRmt and AMPK to extend lifespan. C_LIO_LIGLD-1 represses germline cyc-2.1 translation in the daf-2 rsks-1 mutant. C_LIO_LITranslational regulation of cyc-2.1 and UPRmt contribute to the synergistic longevity of the daf-2 rsks-1 mutant. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/533695v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@9f3ce7org.highwire.dtl.DTLVardef@576930org.highwire.dtl.DTLVardef@bb0c09org.highwire.dtl.DTLVardef@133ce4_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2018

Construction of a germline-specific RNAi tool in C. elegans

Analysis of complex biological functions usually requires tissue-specific genetic manipulations in multicellular organisms. The C. elegans germline plays regulatory roles not only in reproduction, but also in metabolism, stress response and aging. Previous studies have used mutants of rrf-1, which encodes an RNA-directed RNA polymerase, as a germline-specific RNAi tool. However, the rrf-1 mutants showed RNAi efficiency in somatic tissues. Here we constructed a germline-specific RNAi strain by combining an indel mutation of rde-1, which encodes an Argonaute protein that functions cell autonomously to ensure RNAi efficiency, and a single-copy rde-1 transgene driven by the sun-1 germlinespecific promoter. The germline RNAi efficiency and specificity are confirmed by RNAi phenocopy of known mutations, knockdown of GFP reporter expression, as well as quantitative RT-PCR measurement of tissue-specific mRNAs upon RNAi knockdown. The germline-specific RNAi strain shows no obvious deficiencies in reproduction, lipid accumulation, thermo-tolerance and life span compared to wild-type animals. By screening an RNAi sub-library of phosphatase genes, we identified novel regulators of thermo-tolerance. Together, we have created a useful tool that can facilitate the genetic analysis of germline-specific functions in C. elegans.

genetics↗