Fei Teng

Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems

Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems

One-step generation of knockout pigs by zygote injection of CRISPR/Cas system

The pig is an important livestock for food supply and an ideal model for various human diseases. Efficient and precise genetic engineering in pigs holds great promise in agriculture and biomedicine1. Using currently available approach, generating specific gene modifications in pigs requires two steps. First, site-specific nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are used to generate targeted mutations in pig somatic cells. Then the engineered somatic nucleus is used to generate cloned animals using somatic cell nuclear transfer (SCNT) technology2,3. The complex design and generation of ZFNs and TALENs, as well as the technical challenges of SCNT, greatly limit the application of this method.

Genetic modification and screening in rat using haploid embryonic stem cells.

The rat is an important animal model in biomedical research, but practical limitations to genetic manipulation have restricted the application of genetic analysis. Here we report the derivation of rat androgenetic haploid embryonic stem cells (RahESCs) as a tool to facilitate such studies. Our approach is based on removal of the maternal pronucleus from zygotes to generate androgenetic embryos followed by derivation of ESCs. The resulting RahESCs have 21 chromosomes, express pluripotency markers, differentiate into three germ layer cells, and contribute to the germline. Homozygous mutations can be introduced by both large-scale gene trapping and precise gene targeting via homologous recombination or the CRISPR-Cas system. RahESCs can also produce fertile rats after intracytoplasmic injection into oocytes and are therefore able to transmit genetic modifications to offspring. Overall, RahESCs represent a practical tool for functional genetic studies and production of transgenic lines in rat.

One-step generation of p53 gene biallelic mutant Cynomolgus monkey via the CRISPR/Cas system

One-step generation of p53 gene biallelic mutant Cynomolgus monkey via the CRISPR/Cas system

Tex101 is essential for male fertility by affecting sperm migration into the oviduct in mice

Dear Editor, Sperm transport in the female genital tract is physiologically important for mammalian fertilization. The female reproductive system contains multiple natural selective barriers, such as successful uterotubal junction (UTJ) migration and zona pellucida (ZP) binding, to ensure sperm with normal motility and morphology to transmit into oviduct for fertilization (Yanagimachi, 1994; Ikawa et al., 2010). Tex101 is a glycosylphosphatidyl inositol (GPI)-anchored glycoprotein identified as a molecular marker of germ cells (Kurita et al., 2001). Although there have been indications that the malfunction of Tex101 may affect male fertility (Yin et al., 2009), little is known about its exact physiological function and the underlying molecular mechanisms. Recently, a study showed that Tex101 gene knockout sperm were unable to pass through UTJ or bind to ZP, which led to male infertility (Fujihara et al., 2013). Here, we independently generated Tex101 knockout mice and confirmed the infertile phenotype caused by UTJ migration defect. We also found that Tex101 knockout sperm lost the adhesive ability to the surface of female genital tract. Several members of a disintegrin and metalloprotease (ADAM) transmembrane protein family with cell adhesion ability, including ADAM3, ADAM4, ADAM5, and ADAM6, were lost in Tex101 knockout epididymal sperm. These observations may shed new light on the diagnosis of male infertility and development of contraceptive methods in human. High abundant Tex101 protein was only detected in the testis of male mice (Supplementary Figure S1A). To investigate the function of Tex101 in vivo, we generated Tex101 gene knockout mice (Supplementary Figure S1). During the 2-year observation period, neither Tex101 heterozygous mutant (Tex101+/2 ) nor Tex101 homozygous mutant (Tex101 ) mice (over 30 mice per group) showed any overt developmental abnormalities. However, although with normal mating ability, male Tex101 mice could not produce offspring, which confirmed the infertile defect of Tex101 deletion (Supplementary Table S1) (Fujihara et al., 2013). We next characterized the defects of Tex101 sperm causing male infertility. The histology and weight of testis from wildtype (Tex101+/+ ) and Tex101 male mice exhibited no identifiable difference (Supplementary Figure S2). In addition, no difference in sperm count, sperm viability, or motility parameters was observed (Supplementary Table S2). However, none of oocytes from females mated with Tex101 mice was fertilized at 18 h after mating plug formation (Supplementary Figure S3), suggesting that sperm from Tex101 mice were either unable to reach the fertilization place or unable to fertilize the oocytes. We then counted sperm collected from the oviducts of mated females. Large amounts of sperm were found in female mice mated with Tex101+/+ males (323 + 84, n 1⁄4 8), yet no sperm (0, n 1⁄4 24) was recovered from females mated with Tex101 males (Figure 1A). Similarly, sperm were only observed in the UTJ lumen of female mice mated with Tex101+/+ males but not those mated with Tex101 males (Supplementary Figure S4). These results demonstrated that Tex101 sperm were unable to pass through the UTJ of female genital tract. However, Tex101 sperm still fertilized oocytes (Figure 1B) at a lower rate compared with Tex101+/+ sperm (Figure 1C, 40% vs. 58%, P 1⁄4 0.048) in in vitro fertilization (IVF) assays. Moreover, among 24 in-tubal inseminated (ITI) female mice, four were successfully pregnant and produced 12 healthy offspring, indicating that Tex101 sperm were still capable to fertilize oocytes in vivo when the UTJ transportation was avoided (Figure 1D, E, and Supplementary Table S3). In contrast, in intra-uterine insemination (IUI) assays, no offspring was produced in the Tex101 group (Supplementary Table S3), further confirming that the male infertility defect of Tex101 mice was primarily caused by the UTJ migration defect of sperm. We noticed that Tex101 sperm seldom bound to dissected epithelium and ZP in the computer-assisted sperm analysis and IVF experiments. To further assess the membrane adhesive ability of Tex101 sperm, different cells inside the female genital tract, including the epithelium of UTJ and isthmus oviduct, cumulus cells, and oocytes, were dissected out and incubated separately in vitro with Tex101+/+ and Tex101 sperm. After incubation for 30 min, Tex101+/+ sperm adhered to all types of epithelium cells robustly, whereas Tex101 sperm were rarely attached (Figure 1F and G). These results demonstrated that sperm of Tex101 mice had lost their adhesive ability, thus failed to bind to the surface of cells in female genital tract. To investigate the functional mechanisms of Tex101, we used mass spectrometry to characterize the differentially expressed proteins between Tex101+/+ and Tex101 cauda epididymal sperm. A total of 30 proteins were identified with .1.5-fold expression changes, including two ADAM protein family members, ADAM5 and ADAM6 (Supplementary Table S4). Previous studies showed that ADAM3 but not other ADAM proteins played a key role in causing the infertile phenotypes (Ikawa et al., 2010; Fujihara et al., 2013); therefore, we detected the expression of all ADAM family proteins with predominant expression in testis by western blot. All examined proteins had no observable expression difference in testicular sperm between Tex101+/+ and Tex101 mice. However, in cauda doi:10.1093/jmcb/mjt031 Journal of Molecular Cell Biology (2013), 5, 345–347 | 345 Published online August 22, 2013

Immunogenicity and functional evaluation of iPSC-derived organs for transplantation.

Whether physiologically induced pluripotent stem cell (iPSC)-derived organs are immunogenic and can be used for transplantation is unclear. Here, we generated iPSC-derived skin, islet, and heart representing three germ layers of the body through 4n complementation and evaluated their immunogenicity and therapeutic efficacy. Upon transplantation into recipient mice, iPSC-derived skin successfully survived and repaired local tissue wounds. In diabetic mouse models, explanted iPSC-derived islets effectively produced insulin and lowered blood glucose to basal levels. iPSC-derived heart grafts maintained normal beating for more than 3 months in syngeneic recipients. Importantly, no obvious immune rejection responses against iPSC-derived organs were detected long after transplantation. Our study not only demonstrates the fundamental immunogenicity and function of iPSC derivatives, but also provides preclinical evidence to support the feasibility of using iPSC-derived skin, islet, and heart for therapeutic use.

Durable pluripotency and haploidy in epiblast stem cells derived from haploid embryonic stem cells in vitro

Haploid pluripotent stem cells, such as haploid embryonic stem cells (haESCs), facilitate the genetic study of recessive traits. In vitro, fish haESCs maintain haploidy in both undifferentiated and differentiated states, but whether mammalian haESCs can preserve pluripotency in the haploid state has not been tested. Here, we report that mouse haESCs can differentiate in vitro into haploid epiblast stem cells (haEpiSCs), which maintain an intact haploid genome, unlimited self-renewal potential, and durable pluripotency to differentiate into various tissues in vitro and in vivo. Mechanistically, the maintenance of self-renewal potential depends on the Activin/bFGF pathway. We further show that haEpiSCs can differentiate in vitro into haploid progenitor-like cells. When injected into the cytoplasm of an oocyte, androgenetic haEpiSC (ahaEpiSCs) can support embryonic development until midgestation (E12.5). Together, these results demonstrate durable pluripotency in mouse haESCs and haEpiSCs, as well as the valuable potential of using these haploid pluripotent stem cells in high-throughput genetic screening.

A non-invasive method to determine the pluripotent status of stem cells by culture medium microRNA expression detection.

To precisely determine the type and status of cells is an important prerequisite for basic researches and regenerative medicine involving stem cells or differentiated cells. However, the traditional destructive cell status examination methods have many limitations, mainly due to the heterogeneity of cells under the reprogramming or differentiation/trans-differentiation process. Here we present a new method to non-destructively determine the pluripotent level of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), or the types of differentiated cells. The method is achieved by examining the expression profiles of microRNAs (miRNAs) in cell culture medium, which show consistent abundance trend as those of the cellular miRNAs. Therefore, the method enables status examination and afterward application being achieved on the same population of cells, which will greatly facilitate cell reprogramming or differentiation/trans-differentiation related based research and clinical therapy.

MicroRNA-323-3p regulates the activity of polycomb repressive complex 2 (PRC2) via targeting the mRNA of embryonic ectoderm development (Eed) gene in mouse embryonic stem cells.

Background: PRC2 is involved in many biological processes, yet how the function of PRC2 is regulated is largely unknown. Results: The 3′-UTR of Eed mRNA contains miR-323-3p-binding sites. Binding of miR-323-3p to Eed mRNA results in reduced EED protein abundance and cellular H3K27me3 levels in mouse and human cells. Conclusion: miR-323-3p regulates PRC2 activity via targeting Eed. Significance: The work identifies miR-323-3p as a new regulator of PRC2. PRC2 (Polycomb repressive complex 2) mediates epigenetic gene silencing by catalyzing the triple methylation of histone H3 Lys-27 (H3K27me3) to establish a repressive epigenetic state. PRC2 is involved in the regulation of many fundamental biological processes and is especially essential for embryonic stem cells. However, how the formation and function of PRC2 are regulated is largely unknown. Here, we show that a microRNA encoded by the imprinted Dlk1-Dio3 region of mouse chromosome 12, miR-323-3p, targets Eed (embryonic ectoderm development) mRNA, which encodes one of the core components of PRC2, the EED protein. Binding of miR-323-3p to Eed mRNA resulted in reduced EED protein abundance and cellular H3K27me3 levels, indicating decreased PRC2 activity. Such regulation seems to be conserved among mammals, at least between mice and humans. We demonstrate that induced pluripotent stem cells with varied developmental abilities had different miR-323-3p as well as EED and H3K27me3 levels, indicating that miR-323-3p may be involved in the regulation of stem cell pluripotency through affecting PRC2 activity. Mouse embryonic fibroblast cells had much higher miR-323-3p expression and nearly undetectable H3K27me3 levels. These findings identify miR-323-3p as a new regulator for PRC2 and provide a new approach for regulating PRC2 activity via microRNAs.

MicroRNA-494 promotes cancer progression and targets adenomatous polyposis coli in colorectal cancer

BackgroundAberrant activation of the Wnt/β-catenin signaling pathway is frequently observed in colorectal cancer (CRC). β-catenin is the major Wnt signaling pathway effector and inactivation of adenomatous polyposis coli (APC) results in nuclear accumulation of β-catenin. It has been suggested that inactivation of APC plays an important role in activation of the Wnt/β-catenin pathway and in the progression of colorectal tumorigenesis. However, the mechanism through which APC mediates colorectal tumorigenesis is not understood. Increasing evidence suggests that the dysregulation of microRNAs (miRNAs) is involved in colorectal tumorigenesis. Although miR-494 has been reported as being an upregulated miRNA, the interplay between miR-494 and APC-mediated colorectal tumorigenesis progression remains unclear.MethodsThe expression of miR-494 in tissues from patients diagnosed with CRC was analyzed using a microarray and real-time PCR. The effects of miR-494 on cell proliferation and tumorigenesis in CRC cells were analyzed by flow cytometry, colony formation assays, BrdU incorporation assays, and CCK8 assays. The correlation between miR-494 expression and APC expression, as well as the mechanisms by which miR-494 regulates APC in CRC were also addressed.ResultsmiR-494 was significantly upregulated in CRC tissues, and this increase was negatively associated with APC expression. APC was confirmed to be a direct target of miR-494 in CRC. Furthermore, overexpression of miR-494 induced Wnt/β-catenin signaling by targeting APC, thus promoting CRC cell growth.ConclusionsThis study provides novel insights into the role of miR-494 in controlling CRC cell proliferation and tumorigenesis, and identifies miR-494 as a potential prognostic marker and therapeutic target.