Exogenous Small Molecules Research Articles

Antibiotic dialogues: induction of silent biosynthetic gene clusters by exogenous small molecules.

Natural products have traditionally served as a dominant source of therapeutic agents. They are produced by dedicated biosynthetic gene clusters that assemble complex, bioactive molecules from simple precursors. Recent genome sequencing efforts coupled with advances in bioinformatics indicate that the majority of biosynthetic gene clusters are not expressed under normal laboratory conditions. Termed 'silent' or 'cryptic', these gene clusters represent a treasure trove for discovery of novel small molecules, their regulatory circuits and their biosynthetic pathways. In this review, we assess the capacity of exogenous small molecules in activating silent secondary metabolite gene clusters. Several approaches that have been developed are presented, including coculture techniques, ribosome engineering, chromatin remodeling and high-throughput elicitor screens. The rationale, applications and mechanisms attendant to each are discussed. Some general conclusions can be drawn from our analysis: exogenous small molecules comprise a productive avenue for the discovery of cryptic metabolites. Specifically, growth-inhibitory molecules, in some cases clinically used antibiotics, serve as effective inducers of silent biosynthetic gene clusters, suggesting that old antibiotics may be used to find new ones. The involvement of natural antibiotics in modulating secondary metabolism at subinhibitory concentrations suggests that they represent part of the microbial vocabulary through which inter- and intraspecies interactions are mediated.

651. Integration of Dual Signal Inputs Strategies in Novel Chimeric Antigen Receptors to Control the CAR T-Cell Functions

Adoptive immunotherapy using engineered T-cells has emerged as a powerful approach to treat cancer. The potential of this approach relies on the ability to redirect the specificity of T cells through genetic engineering. Novel specificities in T cells have been typically implemented through the genetic transfer of the so-called chimeric antigen receptors (CARs). CARs are synthetic receptors composed of an extracellular targeting moiety and one or more intracytoplasmic signaling domain derived from lymphocyte activation receptors. Present CAR architectures are designed to combine all relevant domains within a single polypeptide, thereby; they combine advantages of MHC unrestricted target recognition to the potent native effector mechanisms of the T cell. Although adoptive transfer of CAR T cells is proven to be an effective cancer therapy, potential adverse effects such as cytokine release syndrome (CRS) and/or the risk of on-target off-tumor targeting are still a major concern.Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. Our ability to engineer synthetic systems in primary T-cells that function as Boolean logic gates responding to multiple inputs would benefit adoptive immunotherapy using engineered T-cells. Exogenous or endogenous environmental signal integration by a modular AND gate may represent an important advancement in improving our control of the safety of the CAR T-cell technology.Here, we describe the development of novel CAR designs that integrate new components directly within the CAR architecture to improve our capacity to spatiotemporally control and switch the CAR T-cells functions between on and off states. In particular, we showed that such a system can be engineered to control the CAR through addition of an exogenous small molecule (Rapamycin or synthetic rapalogs) ultimately inducing the cytolytic properties of the engineered T-cell. Alternatively, properties of the tumor microenvironment can also be used as additional endogenous input to the target antigen recognition. We showed that low oxygen levels can be used to trigger the CAR surface presentation, creating a so called “self-decision making” CAR T-cell.

Color-Coded Super-Resolution Small-Molecule Imaging.

Although the development of super-resolution microscopy dates back to 1994, its applications have been primarily focused on visualizing cellular structures and targets, including proteins, DNA and sugars. We now report on a system that allows both monitoring of the localization of exogenous small molecules in live cells at low resolution and subsequent super-resolution imaging by using stochastic optical reconstruction microscopy (STORM) on fixed cells. This represents a powerful new tool to understand the dynamics of subcellular trafficking associated with the mode and mechanism of action of exogenous small molecules.

Relative Affinities of Positive and Negative Modulators of Heteromeric GABA(A) Receptors for Pseudo-Symmetric Intersubunit Binding Sites

Type-A γ-aminobutyric acid receptors (GABAARs) are pentameric ligand-gated ion channels that are ubiquitous to the central nervous system (CNS) and critical for regulating neuronal excitability. These inhibitory receptors, gated by γ-aminobutyric acid (GABA), can be potentiated and also directly activated by certain exogenous and endogenous lipophilic small molecules. Among the various endogenous modulators are derivatives of cholesterol called neurosteroids; neurosteroids that positively modulate GABAARs can function as natural sedatives, anesthetics, anxiolytics and anti-convulsants. The mechanisms underlying modulation of GABAARs by neurosteroids and exogenous modulators like general anesthetics are still poorly understood, in part due to the location of likely quasi-symmetric interaction sites in the transmembrane domain of the receptor, as well as inconclusive results from mutagenesis experiments. In particular, the extent to which binding modes of positively and negatively modulating neurosteroids overlap is unknown. Here we use molecular dynamics simulations and the thermodynamically rigorous alchemical free energy perturbation technique to rank pseudo-symmetric intersubunit binding modes by affinity for the positively modulating neurosteroid allopregnanolone, the negatively modulating neurosteroid pregnenolone sulfate, and a novel analog with complex dose-response: triiodothyronine (thyroid hormone). Differential rankings of intersubunit sites for positive and negative modulating neurosteroids, triiodothyronine, and the general anesthetics propofol and sevoflurane, are interpreted in the context of their distinctive functional effects.

Long-Term Culture of Porcine Induced Pluripotent Stem-Like Cells Under Feeder-Free Conditions in the Presence of Histone Deacetylase Inhibitors.

The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a complex process that involves significant epigenetic alterations in the reprogrammed cells. Epigenetic modifiers such as histone deacetylase (HDAC) inhibitors have been shown to increase the efficiency of derivation of iPSCs in humans and mice. In this study, we used three HDAC inhibitors, valproic acid, sodium butyrate, and suberoylanilide hydroxamic acid, together with ascorbic acid, for derivation and long-term feeder-free culture of porcine iPS-like cells. In the absence of exogenous growth factors and/or small molecules, these inhibitors were able to maintain the expression of key pluripotency markers, including genes known to be specific for naive pluripotent state in mouse stem cells, for over 60 passages under feeder-free conditions. Surprisingly, the cells became dependent on HDAC inhibitors for the maintenance of proliferation. Moreover, despite showing successful integration into blastocysts upon injection, the cells were unable to undergo normal differentiation in vitro and in vivo in the form of teratomas. Our results suggest that HDAC inhibitors maintain pluripotency gene expression of porcine iPSC-like cells in long-term culture, but prevent lineage specification, requiring further optimization of culture conditions for porcine iPSC derivation.

Nanostructured Silicon-Based Fingerprint Dusting Powders for Enhanced Visualization and Detection by Mass Spectrometry.

Porous silicon microparticles (pSi MPs) functionalized with fluorescent dyes (lissamine and carboxy-5-fluorescein) and intrinsically luminescent pSi MPs were explored as novel fingerprint dusting powders. The versatility of luminescent pSi MPs is demonstrated through time-gated imaging of their long-lived (lifetime>28 μs) near-IR emission, and mass spectrometry analysis of fingerprints dusted with luminescent pSi MPs to provide further information on exogenous small molecules present in latent fingerprints.

139. Controllable Target-Specific Transgene Induction Using an Allosteric trans-Splicing Ribozyme

Gene therapeutic approaches are needed that can achieve inducible, adjustable, and well-controlled expression of therapeutic genes while simultaneously suppressing expression of disease-causing genes for maximization of their efficacy. In this study, we proposed a new approach to address one of the main challenges that current gene therapeutic techniques meet, which is based on group I intron ribozyme. To this end, we established an exogeneously and genetically controllable system through design of an allosteric ribozyme that comprises a Tetrahymena group I-based trans-splicing ribozyme as an active domain for RNA replacement, a small molecule-specific RNAaptamer as a sensor domain, and a communication module as an active transfer domain. This allosteric trans-splicing ribozyme can be used as a system that is able to specifically target, and thus reduce the level of, certain disease-specific RNAs, and simultaneously provides the ability to artificially control transgene expression through RNA replacement in therapeutic targets by using exogenous small molecules to activate the function of the ribozyme. The effectiveness of this approach was assessed by constructing various ribozymes in combination with a theophylline-binding aptamer to identify an allosteric ribozyme whose activity is controlled by theophylline both in vitro and in cell culture assays. Moreover, to confirm allosteric regulation of transgene expression, we constructed adenoviral vectors encoding the ribozymes and validated theophylline-dependent RNA replacement in target RNA-expressing cells. Results demonstrate that an allosteric trans-splicing ribozyme is an applicable RNA-based framework for engineering external ligand-controlled gene expression regulatory systems that exhibit adjustable regulation, design modularity (therefore, broad applicability), and target specificity. Such ribozyme could therefore be useful to monitor and/or modulate fate of any cells (such as cancer cells) using specific endogenous or exogenous cues.

Improved abiotic stress tolerance of bermudagrass by exogenous small molecules

As a widely used warm-season turfgrass in landscapes and golf courses, bermudagrass encounters multiple abiotic stresses during the growth and development. Physiology analysis indicated that abiotic stresses induced the accumulation of ROS and decline of photosynthesis, resulting in increased cell damage and inhibited growth. Proteomic and metabolomic approaches showed that antioxidant enzymes and osmoprotectant contents (sugar, sucrose, dehydrin, proline) were extensively changed under abiotic stress conditions. Exogenous application of small molecules, such as ABA, NO, CaCl2, H2S, polyamine and melatonin, could effectively alleviate damages caused by multiple abiotic stresses, including drought, salt, heat and cold. Based on high through-put RNA seq analysis, genes involved in ROS, transcription factors, hormones, and carbohydrate metabolisms were largely enriched. The data indicated that small molecules induced the accumulation of osmoprotectants and antioxidants, kept cell membrane integrity, increased photosynthesis and kept ion homeostasis, which protected bermudagrass from damages caused by abiotic stresses.

Oligopolyphenylenevinylene-conjugated oligoelectrolyte membrane insertion molecules selectively disrupt cell envelopes of Gram-positive bacteria.

The modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules, such as oligopolyphenylenevinylene-conjugated oligoelectrolyte (OPV-COE) membrane insertion molecules (MIMs), is an emerging biotechnological field. Little is known about the interactions of OPV-COEs with their target, the bacterial envelope. We studied the toxicity of three previously reported OPV-COEs with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV-COEs than Gram-negative bacteria. Transmission electron microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effects of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead, the data support the notion that OPV-COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared from Escherichia coli total lipid extracts alone.

Molecular dynamics of CYP2D6 polymorphisms in the absence and presence of a mechanism-based inactivator reveals changes in local flexibility and dominant substrate access channels.

Cytochrome P450 enzymes (CYPs) represent an important enzyme superfamily involved in metabolism of many endogenous and exogenous small molecules. CYP2D6 is responsible for ∼15% of CYP-mediated drug metabolism and exhibits large phenotypic diversity within CYPs with over 100 different allelic variants. Many of these variants lead to functional changes in enzyme activity and substrate selectivity. Herein, a molecular dynamics comparative analysis of four different variants of CYP2D6 was performed. The comparative analysis included simulations with and without SCH 66712, a ligand that is also a mechanism-based inactivator, in order to investigate the possible structural basis of CYP2D6 inactivation. Analysis of protein stability highlighted significantly altered flexibility in both proximal and distal residues from the variant residues. In the absence of SCH 66712, *34, *17-2, and *17-3 displayed more flexibility than *1, and *53 displayed more rigidity. SCH 66712 binding reversed flexibility in *17-2 and *17-3, through *53 remained largely rigid. Throughout simulations with docked SCH 66712, ligand orientation within the heme-binding pocket was consistent with previously identified sites of metabolism and measured binding energies. Subsequent tunnel analysis of substrate access, egress, and solvent channels displayed varied bottle-neck radii. Taken together, our results indicate that SCH 66712 should inactivate these allelic variants, although varied flexibility and substrate binding-pocket accessibility may alter its interaction abilities.

MALDI Mass Spectrometry Imaging for Visualizing In Situ Metabolism of Endogenous Metabolites and Dietary Phytochemicals.

Understanding the spatial distribution of bioactive small molecules is indispensable for elucidating their biological or pharmaceutical roles. Mass spectrometry imaging (MSI) enables determination of the distribution of ionizable molecules present in tissue sections of whole-body or single heterogeneous organ samples by direct ionization and detection. This emerging technique is now widely used for in situ label-free molecular imaging of endogenous or exogenous small molecules. MSI allows the simultaneous visualization of many types of molecules including a parent molecule and its metabolites. Thus, MSI has received much attention as a potential tool for pathological analysis, understanding pharmaceutical mechanisms, and biomarker discovery. On the other hand, several issues regarding the technical limitations of MSI are as of yet still unresolved. In this review, we describe the capabilities of the latest matrix-assisted laser desorption/ionization (MALDI)-MSI technology for visualizing in situ metabolism of endogenous metabolites or dietary phytochemicals (food factors), and also discuss the technical problems and new challenges, including MALDI matrix selection and metabolite identification, that need to be addressed for effective and widespread application of MSI in the diverse fields of biological, biomedical, and nutraceutical (food functionality) research.

The effects of N-acylhomoserine lactones, β-lactam antibiotics and adenosine on biofilm formation in the multi-β-lactam antibiotic-resistant bacterium Acidovorax sp. strain MR-S7

Bacteria in the natural ecosystem frequently live as adherent communities called biofilms. Some chemical compounds are known to affect biofilm formation. We investigated the effect of exogenous small molecules, N-acylhomoserine lactones (AHLs), β-lactam antibiotics, and adenosine, on biofilm formation in the β-lactam antibiotic-resistant bacterium Acidovorax sp. strain MR-S7. Biofilm formation was induced by the addition of various types of AHL isomers and β-lactam antibiotics, whereas the addition of adenosine strongly interfered with the biofilm formation. A gene (macP) encoding adenosine deaminase (that converts adenosine to inosine controlling intracellular adenosine concentration) was successfully cloned from MR-S7 genome and heterologously expressed in Escherichia coli. The purified MacP protein clearly catalyzed the deamination of adenosine to produce inosine. A transcriptional analysis revealed that biofilm-inducing molecules, an AHL and a β-lactam antibiotic, strongly induced not only biofilm formation but also adenosine deaminase gene expression, suggesting that an elaborate gene regulation network for biofilm formation is present in the β-lactam antibiotic-resistant bacterium studied here.

A brief account of nanoparticle contrast agents for photoacoustic imaging.

Photoacoustic imaging (PAI) is a hybrid, nonionizing modality offering excellent spatial resolution, deep penetration, and high soft tissue contrast. In PAI, signal is generated based on the absorption of laser-generated optical energy by endogenous tissues or exogenous contrast agents leading to acoustic emissions detected by an ultrasound transducer. Research in this area over the years has shown that PAI has the ability to provide both physiological and molecular imaging, which can be viewed alone or used in a hybrid modality fashion to extend the anatomic and hemodynamic sensitivities of clinical ultrasound. PAI may be performed using inherent contrast afforded by light absorbing molecules such as hemoglobin, myoglobin, and melanin or exogenous small molecule contrast agent such as near infrared dyes and porphyrins. However, this review summarizes the potential of exogenous nanoparticle-based agents for PAI applications including contrast based on gold particles, carbon nanotubes, and encapsulated copper compounds.

Stimulation of adipogenesis of adult adipose-derived stem cells using substrates that mimic the stiffness of adipose tissue

Biochemical and biomechanical extracellular matrix (ECM) cues have recently been shown to play a role in stimulating stem cell differentiation towards several lineages, though how they combine to induce adipogenesis has been less well studied. The objective of this study was to recapitulate both the ECM composition and mechanical properties of adipose tissue in vitro to stimulate adipogenesis of human adipose-derived stem cells (ASCs) in the absence of exogenous adipogenic growth factors and small molecules. Adipose specific ECM biochemical cues have been previously shown to influence adipogenic differentiation; however, the ability of biomechanical cues to promote adipogenesis has been less defined. Decellularized human lipoaspirate was used to functionalize polyacrylamide gels of varying stiffness to allow the cells to interact with adipose-specific ECM components. Culturing ASCs on gels that mimicked the native stiffness of adipose tissue (2 kPa) significantly upregulated adipogenic markers, in the absence of exogenous adipogenic growth factors and small molecules. As substrate stiffness increased, the cells became more spread, lost their rounded morphology, and failed to upregulate adipogenic markers. Together these data imply that as with other lineages, mechanical cues are capable of regulating adipogenesis in ASCs.

Contribution of the M1 Transmembrane Helix and Pre-M1 Region to Positive Allosteric Modulation and Gating of N-Methyl-d-Aspartate Receptors

N-methyl-D-aspartate (NMDA) receptors are glutamate-gated ion channels whose function is critical for normal excitatory synaptic transmission in the brain and whose dysfunction has been implicated in several neurologic conditions. NMDA receptor function is subject to extensive allosteric regulation both by endogenous compounds and by exogenous small molecules. Elucidating the structural determinants and mechanism of action by which allosteric regulators control gating will enhance our understanding of NMDA receptor activation and facilitate the development of novel therapeutics. Here, we investigated the structural determinants for (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (CIQ), a GluN2C/2D-selective positive allosteric modulator. We show that CIQ does not bind to the amino-terminal domain of the NMDA receptor and does not share structural determinants with modulators acting at the agonist-binding domain dimer interface or ion channel pore. Rather, we identified critical determinants of CIQ modulation in the region near the first transmembrane helix of GluN2D, including in a putative pre-M1 cuff helix that may influence channel gating. We also show that mutations within the GluN2D pre-M1 region alter open probability of the NMDA receptor. These results suggest a novel site of action for potentiation of NMDA receptors by small molecules and implicate the pre-M1 region in NMDA receptor gating.

Compound Activity Prediction Using Models of Binding Pockets or Ligand Properties in 3D

Transient interactions of endogenous and exogenous small molecules with flexible binding sites in proteins or macromolecular assemblies play a critical role in all biological processes. Current advances in high-resolution protein structure determination, database development, and docking methodology make it possible to design three-dimensional models for prediction of such interactions with increasing accuracy and specificity. Using the data collected in the Pocketome encyclopedia, we here provide an overview of two types of the three-dimensional ligand activity models, pocketbased and ligand property-based, for two important classes of proteins, nuclear and G-protein coupled receptors. For half the targets, the pocket models discriminate actives from property matched decoys with acceptable accuracy (the area under ROC curve, AUC, exceeding 84%) and for about one fifth of the targets with high accuracy (AUC > 95%). The 3D ligand property field models performed better than 95% in half of the cases. The high performance models can already become a basis of activity predictions for new chemicals. Family-wide benchmarking of the models highlights strengths of both approaches and helps identify their inherent bottlenecks and challenges.

Chemical Rescue of the Distal Histidine Mutants of Tryptophan 2,3-Dioxygenase

Tryptophan 2,3-dioxygenase (TDO) is a heme-dependent enzyme that catalyzes the oxidative degradation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK). A highly conserved histidine residue in the distal heme pocket has attracted great attention in the mechanistic studies of TDO. However, a consensus has not been reached regarding whether and how this distal histidine plays a catalytic role after substrate binding. In this study, three mutant proteins, H72S, H72N, and Q73F were generated to investigate the function of the distal histidine residue in Cupriavidus metallidurans TDO (cmTDO). Spectroscopic characterizations, enzymatic kinetic analysis, and chemical rescue assays were employed to study the biochemical properties of the wild-type enzyme and the mutant proteins. Rapid kinetic methods were utilized to explore the molecular basis for the observed stimulation of catalytic activity by 2-methylimidazole in the His72 variants. The results indicate that the distal histidine plays multiple roles in cmTDO. First, His72 contributes to but is not essential for substrate binding. In addition, it shields the heme center from nonproductive binding of exogenous small ligand molecules (i.e., imidazole and its analogs) via steric hindrance. Most importantly, His72 participates in the subsequent chemical catalytic steps after substrate binding possibly by providing H-bonding interactions to the heme-bound oxygen.

The controlled relay of multiple protons required at the active site of nitrogenase

The enzyme nitrogenase, when reducing natural and unnatural substrates, requires large numbers of protons per chemical catalytic cycle. The active face of the catalytic site (the FeMo-cofactor, FeMo-co) is situated in a protein domain which is largely hydrophobic and anhydrous, and incapable of serial provision of multiple protons. Through detailed analysis of the high quality protein crystal structures available the characteristics of a chain of water molecules leading from the protein surface to a key sulfur atom (S3B) of FeMo-co are described. The first half of the water chain from the surface inwards is branched, slightly variable, and able to accommodate exogenous small molecules: this is dubbed the proton bay. The second half, from the proton bay to S3B, is comprised of a single chain of eight hydrogen bonded water molecules. This section is strictly conserved, and is intimately involved in hydrogen bonds with homocitrate, an essential component that chelates Mo. This is the proton wire, and a detailed Grotthuss mechanism for serial translocation of protons through this proton wire to S3B is proposed. This controlled serial proton relay from the protein surface to S3B is an essential component of the intramolecular hydrogenation paradigm for the complete chemical mechanisms of nitrogenase. Each proton reaching S3B, instigated by electron transfer to FeMo-co, becomes a hydrogen atom that migrates to other components of the active face of FeMo-co and to bound substrates and intermediates, allowing subsequent multiple proton transfers along the proton wire. Experiments to test the proposed mechanism of proton supply are suggested. The water chain in nitrogenase is comparable with the purported proton pumping pathway of cytochrome c oxidase.

Use of a coenzyme by the glmS ribozyme-riboswitch suggests primordial expansion of RNA chemistry by small molecules

The glmS ribozyme-riboswitch is the first known example of a naturally occurring catalytic RNA that employs a small molecule as a coenzyme. Binding of glucosamine-6-phosphate (GlcN6P) activates self-cleavage of the bacterial ribozyme, which is part of the mRNA encoding the metabolic enzyme GlcN6P-synthetase. Cleavage leads to negative feedback regulation. GlcN6P binds in the active site of the ribozyme, where its amine could function as a general acid and electrostatic catalyst. The ribozyme is pre-folded but inactive in the absence of GlcN6P, demonstrating it has evolved strict dependence on the exogenous small molecule. The ribozyme showcases the ability of RNA to co-opt non-covalently bound small molecules to expand its chemical repertoire. Analogue studies demonstrate that some molecules other than GlcN6P, such as l-serine (but not d-serine), can function as weak activators. This suggests how coenzyme use by RNA world ribozymes may have led to evolution of proteins. Primordial cofactor-dependent ribozymes may have evolved to bind their cofactors covalently. If amino acids were used as cofactors, this could have driven the evolution of RNA aminoacylation. The ability to make covalently bound peptide coenzymes may have further increased the fitness of such primordial ribozymes, providing a selective pressure for the invention of translation.

Generation of Germline-Competent Rat Induced Pluripotent Stem Cells

BackgroundRecent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs.Methodology/Principal FindingsWe generated riPSCs by transducing three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) into rat somatic cells, followed by culture in the presence of exogenous rat leukemia inhibitory factor (rLIF) and small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. We found that, like rESCs, our riPSCs can contribute to germline transmission. Furthermore we found, by immunostaining of testis from mouse-rat interspecific chimeras with antibody against mouse vasa homolog, that riPSCs can contribute to embryonic development with chimera formation in mice (rat-mouse interspecific chimeras) and to interspecific germlines.Conclusions/SignificanceOur data clearly demonstrate that using only three reprogramming factors (Oct3/4, Klf4, and Sox2) rat somatic cells can be reprogrammed into a ground state. Our generated riPSCs exhibited germline transmission in either rat-rat intraspecific or mouse-rat interspecific chimeras.