Somatic cell nuclear transfer (SCNT) holds great promise for regenerative medicine and agriculture, but its application is severely hampered by low efficiency, primarily attributable to aberrant epigenetic reprogramming. Although embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) have been successfully derived from cloned embryos, an in vitro counterpart of the primitive endoderm (PrE) lineage has remained unavailable. To address this gap, this study reports the first successful establishment of extra-embryonic endoderm stem cell lines (XENs) from mouse SCNT-derived blastocysts (NT-XENs). Under conventional culture conditions, NT-XENs were generated from hybrid B6D2F1 blastocysts at a high efficiency of 55%, statistically comparable to that of fertilization-derived XEN lines (FD-XENs, 50%), whereas derivation from inbred C57BL/6J SCNT-derived blastocysts was markedly lower (12.5%). Immunofluorescence and NanoString multiplex gene expression profiling confirmed that NT-XENs robustly expressed specific marker genes for PrE/XENs (e.g., Gata4, Gata6, and Sox17), while exhibiting negligible or absent expression of pluripotency and trophoblast markers. Based on NanoString assay data, NT-XENs and FD-XENs shared highly similar gene expression patterns, yet also exhibited some nonnegligible differences, exemplified by the differentially expressed genes (DEGs) Pecam1, Gtl2, Thbd, and Xlr3b. These differences raise a preliminary hypothesis that the NT-XENs might exhibit a slight transcriptional propensity toward a more differentiated state, and potentially reflect lingering traces of SCNT-associated epigenetic errors, such as localized dysregulation of imprinted genes and X-linked transcripts. In summary, this study successfully establishes NT-XEN cell lines, providing a valuable in vitro model for investigating the reprogramming scenarios of PrE lineage in SCNT and the mechanisms underlying developmental failure of cloned embryos.

Tetraploidy occurs infrequently in mammals but remains widespread in amphibians. Blastocyst complementation using xenogeneic transplantation of tetraploid embryonic stem cells (4N-ESCs) represents a promising approach to mitigate organ shortages, yet robust generation of fully reconstituted organs in mammalian hosts remains elusive. In this study, CRISPR/Cas9, the Cre-LoxP system, and blastocyst complementation were combined to generate tetraploid mouse liver, heart, and pancreatic tissues. 4N-ESCs (tdTomato-labeled) were established and shown to maintain stable pluripotency and tetraploidy, as confirmed by karyotyping and immunofluorescence analyses. Subsequently, these cells were microinjected into Hhexand Pdx1-deficient blastocysts and Nkx2.5 lineage-ablated blastocysts, which were engineered to lack relevant organ-forming lineages. Tetraploid pups exhibited significantly reduced body mass and organ mass (liver and heart) relative to diploid controls (P<0.05). Fluorescence-activated cell sorting demonstrated a significant 4N-ESC (tdTomato-labeled) contribution within tetraploid organs (4N population) at E18.5, with tdTomato-positive fractions reaching 84.3% of hepatic cells, 67.8% of cardiac cells, and 73.4% of pancreatic cells. Single-cell transcriptome sequencing further revealed that tetraploidy markedly altered developmental trajectories and differentiation programs in liver and heart tissues, and 4N-ESCs showed preferential integration into tetraploid liver and heart with a substantial contribution to pancreatic regeneration. Collectively, these findings support the feasibility of 4N- ESC-based blastocyst complementation for human organ regeneration and establish a framework for developing strategies to alleviate organ shortages in clinical settings.

Semi-cloning technology has made a series of advances in studying gene function and building disease models. Maternally semi-cloned mice ( MSCM ) have completely identical maternal genome. MSCM are typically produced by injecting sperm into oocytes whose spindles have been replaced by parthenogenetic haploid embryonic stem cells ( PG-haESCs ), resulting in an extremely low birth rate. However, injecting parthenogenetic double haploid ESCs ( PG-DhESCs ) into Prdm14 knockout embryos generates chimeric mice whose germ cells originate from the PG-DhESCs and produce genetically identical oocytes. The offsprings of those chimeric mice, named MSCM, have completely identical maternal genome, mitochondrial genome of the same origin, and an equal proportions of male and female,

With 296 million chronically infected individuals worldwide, hepatitis B virus (HBV) causes a major health burden. The major challenge to cure HBV infection lies in the fact that the source of persistence infection, viral episomal covalently closed circular DNA (cccDNA), could not be targeted. In addition, HBV DNA integration, although normally results in replication-incompetent transcripts, considered as oncogenic. Though several studies evaluated the potential of gene-editing approaches to target HBV, previous in vivo studies have been of limited relevance to authentic HBV infection, as the models do not contain HBV cccDNA or feature a complete HBV replication cycle under competent host immune system. In this study, we evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. Our study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection.

Understanding the mechanisms underlying phenotypic innovation is a key goal of comparative genomic studies. Here, we investigated the evolutionary landscape of lineage-specific accelerated regions (LinARs) across 49 primate species. Genomic comparison with dense taxa sampling of primate species significantly improved LinAR detection accuracy and revealed many novel human LinARs associated with brain development or disease. Our study also yielded detailed maps of LinARs in other primate lineages that may have influenced lineage-specific phenotypic innovation and adaptation. Functional experimentation identified gibbon LinARs, which could have participated in the developmental regulation of their unique limb structures, whereas some LinARs in the Colobinae were associated with metabolite detoxification which may have been adaptive in relation to their leaf-eating diet. Overall, our study broadens knowledge of the functional roles of LinARs in primate evolution.

Although the continual expansion of the brain during primate evolution accounts for our enhanced cognitive capabilities, the drivers of brain evolution have scarcely been explored in these ancestral nodes. Here, we performed large-scale comparative genomic, transcriptomic, and epigenomic analyses to investigate the evolutionary alterations acquired by brain genes and provide comprehensive listings of innovatory genetic elements along the evolutionary path from ancestral primates to human. The regulatory sequences associated with brain-expressed genes experienced rapid change, particularly in the ancestor of the Simiiformes. Extensive comparisons of single-cell and bulk transcriptomic data between primate and nonprimate brains revealed that these regulatory sequences may drive the high expression of certain genes in primate brains. Employing in utero electroporation into mouse embryonic cortex, we show that the primate-specific brain-biased gene BMP7 was recruited, probably in the ancestor of the Simiiformes, to regulate neuronal proliferation in the primate ventricular zone. Our study provides a comprehensive listing of genes and regulatory changes along the brain evolution lineage of ancestral primates leading to human. These data should be invaluable for future functional studies that will deepen our understanding not only of the genetic basis of human brain evolution but also of inherited disease.

DEAR EDITOR, The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)pandemic remains an important global public health issue.In this study,we unexpectedly found that wild-type Sprague-Dawley(SD)rats can be infected with the SARS-CoV-2 prototype.Our results showed direct experimental evidence of the infectivity of SARS-CoV-2 infection,subsequent pathogenicity,and protection against reinfection in rats.

Totipotency emerges in early embryogenesis, but its molecular underpinnings remain poorly characterized. In the present study, we employed DNA fiber analysis to investigate how pluripotent stem cells are reprogrammed into totipotent-like 2-cell-like cells (2CLCs). We show that totipotent cells of the early mouse embryo have slow DNA replication fork speed and that 2CLCs recapitulate this feature, suggesting that fork speed underlies the transition to a totipotent-like state. 2CLCs emerge concomitant with DNA replication and display changes in replication timing (RT), particularly during the early S-phase. RT changes occur prior to 2CLC emergence, suggesting that RT may predispose to gene expression changes and consequent reprogramming of cell fate. Slowing down replication fork speed experimentally induces 2CLCs. In vivo, slowing fork speed improves the reprogramming efficiency of somatic cell nuclear transfer. Our data suggest that fork speed regulates cellular plasticity and that remodeling of replication features leads to changes in cell fate and reprogramming.

SUMMARY Extraembryonic endoderm stem (XEN) cell lines can be derived and maintained in vitro and reflect the primitive endoderm cell lineage. SOX17 is thought to be required for the derivation and maintenance of mouse XEN cell lines. Here we have re-evaluated this requirement for SOX17. We derived multiple SOX17-deficient XEN cell lines from preimplantation embryos of a SOX17-Cre knockout strain and chemically converted multiple SOX17-deficient embryonic stem cell lines into XEN cell lines by transient culturing with retinoic acid and Activin A. We confirmed the XEN profile of SOX17-deficient cell lines by immunofluorescence with various markers, by NanoString gene expression analyses, and by their contribution to the extraembryonic endoderm of chimeric embryos produced by injecting these cells into blastocysts. Thus, SOX17 is not required for the derivation and maintenance of XEN cell lines.

Mouse extraembryonic endoderm stem (XEN) cell lines can be derived from preimplantation embryos (pre-XEN) and postimplantation embryos (post-XEN). XEN cells share a gene expression profile and cell lineage potential with primitive endoderm (PrE) blastocysts. However, the cellular origin of XEN cells in embryos remains unclear. Here, we report that post-XEN cell lines are derived both from the extraembryonic endoderm and epiblasts of postimplantation embryos and that pre-XEN cell lines are derived both from PrE and epiblasts of blastocysts. Our strategy consisted of deriving post-XEN cells from clumps of epiblasts, parietal endoderm (PE) and visceral endoderm (VE) and deriving pre-XEN cell lines from single PrE and single epiblasts of blastocysts. Thus, XEN cell lines in the mouse embryo originate not only from PrE and PrE-derived lineages but also from epiblast and epiblast-derived lineages of blastocysts and postimplantation embryos. ### Competing Interest Statement The authors have declared no competing interest.

In the mouse, most mature olfactory sensory neurons (OSNs) express one allele of one gene from the repertoire of ~1100 odorant receptor (OR) genes, which encode G-protein coupled receptors (GPCRs). Axons of OSNs that express a given OR coalesce into homogeneous glomeruli, which reside at conserved positions in the olfactory bulb. ORs are intimately involved in ensuring the expression of one OR per OSN and the coalescence of OSN axons into glomeruli. But the mechanisms whereby ORs accomplish these diverse functions remain poorly understood. An experimental approach that has been informative is to substitute an OR genetically with another GPCR that is normally not expressed in OSNs, in order to determine in which aspects this GPCR can serve as surrogate OR in mouse OSNs. Thus far only the β2-adrenergic receptor (β2AR, Ardb2) has been shown to be able to serve as surrogate OR in OSNs; the β2AR could substitute for the M71 OR in all aspects examined. Can other non-olfactory GPCRs function equally well as surrogate ORs in OSNs? Here, we have generated and characterized two novel gene-targeted mouse strains in which the mouse melanocortin 4 receptor (Mc4r) or the mouse dopamine receptor D1 (Drd1a) is coexpressed with tauGFP in OSNs that express the OR locus M71. These alleles and strains are abbreviated as Mc4r → M71-GFP and Drd1a → M71-GFP. We detected strong Mc4r or Drd1a immunoreactivity in axons and dendritic knobs and cilia of OSNs that express Mc4r or Drd1a from the M71 locus. These OSNs responded physiologically to cognate agonists for Mc4r (Ro27-3225) or Drd1a (SKF81297), and not to the M71 ligand acetophenone. Axons of OSNs expressing Mc4r → M71-GFP coalesced into glomeruli. Axons of OSNs expressing Drd1a → M71-GFP converged onto restricted areas of the olfactory bulb but did not coalesce into glomeruli. Thus, OR functions in OSNs can be substituted by Mc4r or Drd1a, but not as well as by β2AR. We attribute the weak performance of Drd1a as surrogate OR to poor OSN maturation.

Extraembryonic endoderm stem (XEN) cell lines can be derived and maintained in vitro and reflect the primitive endoderm lineage. Platelet-derived growth factor receptor alpha (PDGFRA) is thought to be essential for the derivation and maintenance of mouse XEN cell lines. Here, we have re-evaluated this requirement for PDGFRA. We derived multiple PDGFRA-deficient XEN cell lines from postimplantation and preimplantation embryos of a PDGFRA-GFP knockout strain. We also converted PDGFRA-deficient embryonic stem cell lines into XEN cell lines chemically by transient culturing with retinoic acid and Activin A. We confirmed the XEN profile of our 12 PDGFRA-deficient cell lines by immunofluorescence with various markers, by NanoString gene expression analyses, and by their contribution to the extraembryonic endoderm of chimeric embryos produced by injecting these cells into blastocysts. Thus, PDGFRA is not essential for the derivation and maintenance of XEN cell lines.

SummaryGene targeting in embryonic stem (ES) cells remains best practice for introducing complex mutations into the mouse germline. One aspect in this multistep process that has not been streamlined with regard to the logistics and ethics of mouse breeding is the efficiency of germline transmission: the transmission of the ES cell‐derived genome through the germline of chimeras to their offspring. A method whereby male chimeras transmit exclusively the genome of the injected ES cells to their offspring has been developed. The new technology, referred to as goGermline, entails injecting ES cells into blastocysts produced by superovulated homozygous Tsc22d3 floxed females mated with homozygous ROSA26‐Cre males. This cross produces males that are sterile due to a complete cell‐autonomous defect in spermatogenesis. The resulting male chimeras can be sterile but when fertile, they transmit the ES cell‐derived genome to 100% of their offspring. The method was validated extensively and in two laboratories for gene‐targeted ES clones that were derived from the commonly used parental ES cell lines Bruce4, E14, and JM8A3. The complete elimination of the collateral birth of undesired, non‐ES cell‐derived offspring in goGermline technology fulfills the reduction imperative of the 3R principle of humane experimental technique with animals. genesis 54:326–333, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

Various types of stem cell lines have been derived from preimplantation or postimplantation mouse embryos: embryonic stem cell lines, epiblast stem cell lines, and trophoblast stem cell lines. It is not known if extraembryonic endoderm stem (XEN) cell lines can be derived from postimplantation mouse embryos. Here, we report the derivation of 77 XEN cell lines from 85 postimplantation embryos at embryonic day E5.5 or E6.5, in parallel to the derivation of 41 XEN lines from 69 preimplantation embryos at the blastocyst stage. We attain a success rate of 100% of XEN cell line derivation with our E5.5 whole-embryo and E6.5 disaggregated-embryo methods. Immunofluorescence and NanoString gene expression analyses indicate that the XEN cell lines that we derived from postimplantation embryos (post-XEN) are very similar to the XEN cell lines that we derived from preimplantation embryos (pre-XEN) using a conventional method. After injection into blastocysts, post-XEN cells contribute to extraembryonic endoderm in chimeras at E6.5 and E7.5.

Isolation of different cells from adipose tissue was performed according to the previous reports (Rodeheffer et al., 2008; Sugii et al., 2010). Briefly, the inguinal fat pads of adult male B6D2F1 (8-12 weeks of age) mice were harvested, washed several times with phosphate-buffered saline (PBS) and excised into small pieces. The tissues were digested with 0.1% type I collagenase (Sigma) for 50 min at 37°C shaking, followed by adding equal volume of Dulbecco’s modified Eagle’s medium (DMEM; Hyclone) with 10% fetal bovine serum (FBS; Gibico) to neutralize enzyme activity. The cell suspensions were centrifuged at 400 g for 8 min and the supernatant was removed. The pelleted cells, so-called SVF cells, were suspended with PBS containing 2% FBS and were incubated with antibodies. Antibodies used in this study were purchased from eBioscience unless otherwise stated, including CD45-APC-Cy7, Terr119-FITC, CD31-biotin (BD Biosciences), PE-Texas Red (BD Biosciences), CD140a-PE, CD140b-PE, CD105-PE, CD13-FITC, Sca-1-PE and CD34-APC. Antibody incubations were performed on ice for 20 min. Samples were sorted on a BD FACSAria II cell sorter and analyzed on a BD Calibur flow cytometer, each equipped with BD FACSDiva Software. The cells were separated on the basis of the cell-surface markers indicated. Sorted Lin cells and CD45 + cells were suspended in HEPES-buffered CZB (HCZB; Sigma) medium with 2% polyvinylpyrrolidone (PVP; Sigma) for nuclear transfer (NT).

Self-renewal and pluripotency are hallmarks of embryonic stem cells (ESCs). However, the signaling pathways that trigger their transition from self-renewal to differentiation remain elusive. Here, we report that calcineurin-NFAT signaling is both necessary and sufficient to switch ESCs from an undifferentiated state to lineage-specific cells and that the inhibition of this pathway can maintain long-term ESC self-renewal independent of leukemia inhibitory factor. Mechanistically, this pathway converges with the Erk1/2 pathway to regulate Src expression and promote the epithelial-mesenchymal transition (EMT), a process required for lineage specification in response to differentiation stimuli. Furthermore, calcineurin-NFAT signaling is activated when the earliest differentiation event occurs in mouse embryos, and its inhibition disrupts extraembryonic lineage development. Collectively, our results demonstrate that the NFAT and Erk1/2 cascades form a signaling switch for early lineage segregation in mouse ESCs and provide significant insights into the regulation of the balance between ESC self-renewal and early lineage specification.

Dear Editor, Recent studies show that induced pluripo-tent stem cells (iPSCs) generated through ectopic expression of transcription factors retain an epigenetic memory of their original somatic cells (Kim et al., 2010; Polo et al., 2010) or aberrant silencing of a single imprinted gene cluster (Liu et al., 2010; Stadtfeld et al., 2010), which affects their developmental and differentiation potentials. In contrast, nuclear transfer can more faithfully reprogramme somatic cells into embryonic stem (ES) cells (nuclear transfer ES cells, ntESCs) 2006). However, it is still controversial whether reprogramming method per se determines the pluripotency of resulting cells. Here, using genetically identical donor cells, we generated three kinds of mouse reprogrammed cells: iPSCs, ntESCs, and iPSC-nt-ESCs, after successively reprogramming of iPSCs by nuclear transfer. We found that ntESCs had better developmental potential compared with iPSCs, and following nuclear transfer can not rescue, but deteriorate the developmental deficiency of iPSCs, resulting in the worst developmental ability in iPSC-nt-ESCs. In order to minimize genetic variations among different reprogrammed cells, we established a genetically homogenous secondary reprogramming system, in which mouse embryonic fibroblasts (MEFs) carrying doxycycline (Dox)-inducible lentiviruses expressing Oct4, Sox2, Klf4, and c-Myc (OSKM-MEFs) were isolated (Huang et al., 2009) and used as donors for different reprogramming experiments. To generate secondary iPS cells, OSKM-MEFs were cultured in mouse embryonic stem cell medium supplemented with Dox, which induced the expression of transgenes and initiated the reprogramming process. After exposing OSKM-MEFs to Dox for 18 days, iPS cell colonies were expanded in the absence of Dox and four iPS cell lines were established from MEF-1 and MEF-2 (iPSC-1 and iPSC-2 generated from MEF-1; iPSC-3 and iPSC-4 from MEF-2; Figure 1A; Supplementary Figure S1A). All iPS cell lines showed an ES-like morphology , exhibited alkaline phosphatase (AP) activity and expressed the pluripotent markers Oct4, Sox2, and SSEA-1 (see Supplementary Figure S1B). Bisulphite sequencing analysis of endogenous Oct4 promoter of iPS cell nuclei revealed that epigenetic state of the somatic cells had been reprogrammed (Supplementary Figure S1C). These results indicated that all iPSCs had activated their endo-genous pluripotency core transcriptional network. To generate genetic identical ntESCs, we performed nuclear transfer (Yang et al., 2010) using the same MEFs as nucleus donors (Figure 1A). From 526 oocytes successfully reconstructed with the nuclei of MEF-1 and MEF-2, we cloned blastocysts and established a set of 12 ntES cell lines (Supplementary Table S1). Next, to test whether successively reprogramming by nuclear …