Genome-edited rabbits:Unleashing the potential of a promising experimental animal model across diverse diseases

Animal models are extensively used in all aspects of biomedical research,with substantial contributions to our understanding of diseases,the development of pharmaceuticals,and the exploration of gene functions.The field of genome modification in rabbits has progressed slowly.However,recent advancements,particularly in CRISPR/Cas9-related technologies,have catalyzed the successful development of various genome-edited rabbit models to mimic diverse diseases,including cardiovascular disorders,immunodeficiencies,agingrelated ailments,neurological diseases,and ophthalmic pathologies.These models hold great promise in advancing biomedical research due to their closer physiological and biochemical resemblance to humans compared to mice.This review aims to summarize the novel gene-editing approaches currently available for rabbits and present the applications and prospects of such models in biomedicine,underscoring their impact and future potential in translational medicine.

A booming field of large animal model research

Animal models are integral to the study of fundamental biological processes and the etiology of human diseases.Small animal models,especially those involving mice,have yielded abundant and significant insights,greatly enhancing our understanding of biological phenomena and disease mechanisms.

Niclosamide,an old antihelminthic agent,demonstrates antitumor activity by blocking multiple signaling pathways of cancer stem cells

Niclosamide,an oral antihelminthic drug,has been used to treat tapeworm infection for about 50 years.Niclosamide is also used as a molluscicide for water treatment in schistosomiasis control programs.Recently,several groups have independently discovered that niclosamide is also active against cancer cells,but its precise mechanism of antitumor action is not fully understood.Evidence supports that niclosamide targets multiple signaling pathways (NF-κB,Wnt/β-catenin,Notch,ROS,mTORC1,and Stat3),most of which are closely involved with cancer stem cells.The exciting advances in elucidating the antitumor activity and the molecular targets of this drug will be discussed.A method for synthesizing a phosphate pro-drug of niclosamide is provided.Given its potential antitumor activity,clinical trials for niclosamide and its derivatives are warranted for cancer treatment.

Bioreductive prodrugs as cancer therapeutics:targeting tumor hypoxia

Hypoxia, a state of low oxygen, is a common feature of solid tumors and is associated with disease progression as well as resistance to radiotherapy and certain chemotherapeutic drugs. Hypoxic regions in tumors, therefore, represent attractive targets for cancer therapy. To date, five distinct classes of bioreactive prodrugs have been developed to target hypoxic cells in solid tumors. These hypoxia-activated prodrugs, including nitro compounds, N-oxides, quinones, and metal complexes, generally share a common mechanism of activation whereby they are reduced by intracellular oxidoreductases in an oxygensensitive manner to form cytotoxins. Several examples including PR-104, TH-302, and EO9 are currently undergoing phase II and phase III clinical evaluation. In this review, we discuss the nature of tumor hypoxia as a therapeutic target, focusing on the development of bioreductive prodrugs. We also describe the current knowledge of how each prodrug class is activated and detail the clinical progress of leading examples.

Novel rhino-like SHJH^(hr) mice with thyroid dysfunction

A new Hairless(Hr)gene mutant mouse line(SHJH^(hr))was identified,which showed hairless skin in adult individuals as reported in rhino mice.Through Hr gene mutant identification with polymerase chain reaction(PCR)amplification and sequencing,seven mutants were identified,including nonsense mutant site 2134C→T(R467X),which produced a truncated Hr protein.Metabolic activity and heart rate were measured using a metabolic cage and blood pressure instrument,respectively.The SHJH^(hr) mice showed a strong metabolic rate,high heart rate,and low blood pressure.Histological analysis of the thyroid gland of SHJH^(hr) mice showed abnormal follicular structure and hypertrophic thyrocytes.Compared to ICR mice,thyroid function in 4-month-old SHJH^(hr) mice showed lower thyroid stimulating hormone(TSH)levels,and in 9-month-old SHJH^(hr) mice showed significantly higher TSH and thyroid hormone levels.These data indicate that SHJH^(hr) mice may be in a hyperthyroid state with increasing age.Thus,based on the above results,we successfully established a novel mouse model with thyroid dysfunction.

Multiple gene modifications of pigs for overcoming obstacles of xenotransplantation

Xenotransplantation,involving animal organ transplantation into humans to address the human organ shortage,has been studied since the 17th century.Early attempts to obtain organs from animals such as goats,dogs,and non-human primates proved unsuccessful.In the 1990s,scientists agreed that pigs were the most suitable donor animals for xenotransplantation.However,immune rejection between pig and human has hindered the application.To overcome these challenges,researchers developed genetically modified pigs that deactivate xenoreactive antigen genes and express human protective genes.These advances extended xenograft survival from days to years in non-human primates,resulting in the first human heart xenotransplant trial.Using genetically engineered pigs for the organ shortage is promising.This review provides an overview of potential incompatibilities of immunogenicity and functional proteins related to xenotransplantation between humans and pigs.Furthermore,it elucidates possible approaches for multiplex gene modification to breed better-humanized pigs for clinical xenotransplantation.

Integration of Computational Analysis and Spatial Transcriptomics in Single-cell Studies

Recent advances of single-cell transcriptomics technologies and allied computational methodologies have revolutionized molecular cell biology.Meanwhile,pioneering explorations in spatial transcriptomics have opened up avenues to address fundamental biological questions in health and diseases.Here,we review the technical attributes of single-cell RNA sequencing and spatial transcriptomics,and the core concepts of computational data analysis.We further highlight the challenges in the application of data integration methodologies and the interpretation of the biological context of the findings.

Generation of pigs with humanized type Ⅱ collagen by precise human COL2A1 gene knock-in

Osteoarthritis(OA)is the most common chronic disease,characterized by progressive cartilage breakdown,subchondral bone sclerosis,and aberrant bone outgrowth(Yucesoy et al.,2015;Hussain et al.,2016).OA is one of the leading causes of cartilage damage.Patients with severe cartilage damage require transplantation of articular cartilage to improve their quality of life.Type Ⅱ collagen is a major component of articular cartilage and intervertebral discs and plays an important role in the structure and strength of connective tissues that support muscles and joints(Byers,1994).

乳酸化修饰参与调控DNA损伤修复和癌细胞化疗敏感性

The phenomenon in which cells prefer glycolysis to oxidative phosphorylation with increased lactate production is known as the Warburg effect and is found prevalent in cancer cells and pluripotent stem cells [1]. In addition to being an intermediate metabolite of glycolysis, lactate has been reported as a signal involved in multiple important biological processes, such as innate immunity [2], the cell cycle [3], hippocampal neurogenesis [4].

Transposable elements at the center of the crossroads between embryogenesis, embryonic stem cells, reprogramming,and long non-coding RNAs

Transposable elements(TEs) are mobile genomic sequences of DNA capable of autonomous and nonautonomous duplication. TEs have been highly successful,and nearly half of the human genome now consists of various families of TEs. Originally thought to be non-functional,these elements have been co-opted by animal genomes to perform a variety of physiological functions ranging from TE-derived proteins acting directly in normal biological functions, to innovations in transcription factor logic and influence on epigenetic control of gene expression. During embryonic development, when the genome is epigenetically reprogrammed and DNA-demethylated, TEs are released from repression and show embryonic stage-specific expression, and in human and mouse embryos, intact TEderived endogenous viral particles can even be detected. Asimilar process occurs during the reprogramming of somatic cells to pluripotent cells: When the somatic DNA is demethylated, TEs are released from repression. In embryonic stem cells(ESCs), where DNA is hypomethylated, an elaborate system of epigenetic control is employed to suppress TEs, a system that often overlaps with normal epigenetic control of ESC gene expression. Finally, many long non-coding RNAs(lnc RNAs) involved in normal ESC function and those assisting or impairing reprogramming contain multiple TEs in their RNA. These TEs may act as regulatory units to recruit RNA-binding proteins and epigenetic modifiers. This review covers how TEs are interlinked with the epigenetic machinery and lnc RNAs, and how these links influence each other to modulate aspects of ESCs,embryogenesis, and somatic cell reprogramming.

Role of Lef1 in sustaining self-renewal in mouse embryonic stem cells

Embryonic stem cells （ESCs） can self-renew indefinitely while maintaining the ability to generate all three germ-layer derivatives.Despite the importance of ESCs in developmental biology and their potential impact on regenerative medicine,the molecular mechanisms controlling ESC behavior are incompletely understood.Previously,activation of the canonical Wnt signaling pathway has been shown to contribute to mouse ESC self-renewal.Here we report that ectopic expression of Lef1,a component of the Wnt signaling pathway,has a positive effect on the self-renewal of mouse ESCs.Lef1 up-regulates Oct4 promoter activity and physically interacts with Nanog,two key components of the ESC pluripotency machinery.Moreover,siRNA for Lef1 induced mouse ESC differentiation.Our results thus suggest that in response to Wnt signaling Lef1 binds to stabilized β-catenin and helps maintain the undifferentiated status of ESCs through modulation of Oct4 and Nanog.

In vitro induction of mouse meningeal-derived iPS cells into neural-like cells

Previous research has shown that mouse embryonic stem (ES) cells can be induced to form neural cells in adherent monocultures.In this study,pluripotent stem (iPS) C5 cells derived from meningeal membranes were converted successfully into neural-like cells using the same protocol generally used for ES cells.Meningeal-iPS C5 cells were induced to express neural markers Sox1,Sox3,Pax6,Nestin and Tuj1 and to reduce the expression of ES markers Oct4 and Nanog during neural differentiation,and can be differentiated into Pax6 and Nestin positive neural progenitors,and further into neuronal,astrocytic,and oligodendrocytic cells.In vitro differentiation of iPS cells into patient-specific neural cells could serve as a model to study mechanisms of genetic diseases and develop promising candidates for therapeutic applications in dysfunctional or aging neural tissues.Meningeal cells express a high level of the embryonic master regulator Sox2,allowing them to be reprogrammed into iPS cells more easily than other somatic cells.

Fargeted elimination of mutant mitochondria DNA in MELAS-iPSCs by mitoTALENs

Mitochondrial diseases are maternally inherited hetero- geneous disorders that are primarily caused by mitochondrial DNA （mtDNA） mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruc- tion of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells （MiPSCs） that harbored a high proportion of m.3243A〉G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes （MELAS）. We engineered mitochondrial-targeted transcription activator-like effector nucleases （mitoTALENs） and successfully eliminated the m.3243A〉G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Further- more, we successfully achieved reduction in the human m.3243A〉G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.

Comments on ‘Molecular architecture of lineage allocation and tissue organization in early mouse embryo’

Single-cell RNA-seq,with its capability to align cells of continuously changed status by pseudo-time reconstruction,has greatly revolutionized the understanding of cell fate transition during embryo development(Shapiroetal.,2013;Hoppeetal.,2014).

A tunable, rapid, and precise drug control of protein expression by combining transcriptional and post-translational regulation systems

Rapid,precise,and tunable regulation of protein abundance would be significantly useful in a variety of biotechnologies and biomedical applications.Here,we describe a system that allows tunable and rapid drug control of gene expression for either gene activation or inactivation in mammalian cells.We construct the system by coupling Tet-on 3 G and small molecule-assisted shutoff systems,which can respectively induce transcriptional activation and protein degradation in the presence of corresponding small molecules.This dual-input drug inducer regulation system facilitates a bidirectional control of gene expression.The gene of interest can be precisely controlled by dual small molecules in a broad dynamic range of expression from overexpression to complete silence,allowing gene function study in a comprehensive expression profile.Our results reveal that the bidirectional control system enables sensitive dosage-and time-dependent regulation for either turn-on or shutoff of gene expression.We also apply this system for inducible genome editing and gene activation mediated by clustered regularly interspaced short palindromic repeats.The system provides an integrated platform for studying multiple biological processes by manipulating gene expression in a more flexible way.

Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells

Background:Tooth is vital not only for a good smile,but also good health.Yet,we lose tooth regularly due to accidents or diseases.An ideal solution to this problem is to regenerate tooth with patients’own cells.Here we describe the generation of tooth-like structures from integration-free human urine induced pluripotent stem cells(ifhU-iPSCs).Results:We first differentiated ifhU-iPSCs to epithelial sheets,which were then recombined with E14.5 mouse dental mesenchymes.Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30%for 8 different iPSC lines,comparable to H1 hESC.We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures,possessing physical properties such as elastic modulus and hardness found in the regular human tooth.Conclusion:Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.

Generation of gene-target dogs using CRISPR/Cas9 system

Dear Editor,Dogs(Canis familiaris)serve as human companions and are raised to herd livestock,aid hunters,guard homes,perform police and rescue work,and guide the blind.Dogs exhibit close similarities to humans in terms of metabolic,physiological,and anatomical characteristics,and thus are ideal genetic and clinical models to study human diseases(Tsai et al.,2007).Gene target technology is a powerful tool to create new strains of animals with favorable traits.However,thus far,gene-target dogs have not been developed due to their unique species-specific reproductive characteristics,which limits the applications of dogs especially in the field of biomedical research.Recently,clustered regularly interspaced short palindromic repeats(CRISPRs)/CRISPR-associated(Cas)9 system was applied to edit specific genes with a high efficiency(Cong et al.,2013;Mali et al.,2013).Here we attempt to explore the feasibility of producing gene knockout(KO)dogs by using this technology.Beagle dog,the most widely used breed in biomedical research,was used as our animal model.Myostatin(MSTN)was chosen as the first gene of interest.

Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process.Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline.Previous pig models with tetracycline regulatory elements were generated through random integration.This process often resulted in uncertain expression and unpredictable phenotypes,thus hindering their applications.Here,by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus,respectively,a double knock-in reporter pig model was generated.We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo.Two att P sites were arranged to flank the td Tomato to switch reporter gene.Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos.To display the flexible application of this system,we generated a pig strain with Dox-inducing h KRASexpression through phiC31 integrase-mediated cassette exchange.After eight months of Dox administration,squamous cell carcinoma developed in the nose,mouth,and scrotum,which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis.Overall,the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation

Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs)into mouse blastocysts with deficiency of Flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS)of Flk-1^EGFP/ECFP rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.