Efficient Generation of Germ Line Transmitting Chimeras from C57BL/6N ES Cells by Aggregation with Outbred Host Embryos

Genetically modified mouse strains derived from embryonic stem (ES) cells have become essential tools for functional genomics and biomedical research. Large scale mutagenesis projects are producing libraries of mutant C57BL/6 (B6) ES cells to enable the functional annotation of every gene of the mouse genome. To realize the utility of these resources, efficient and accessible methods of generating mutant mice from these ES cells are necessary. Here, we describe a combination of ICR morula aggregation and a chemically-defined culture medium with widely available and accessible components for the high efficiency generation of germline transmitting chimeras from C57BL/6N ES cells. Together these methods will ease the access of the broader biomedical research community to the publicly available B6 ES cell resources

Induced Pluripotent Stem Cell Lines Derived from Equine Fibroblasts

The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapies before moving into the human clinical setting. In this study, we report the generation of iPS cells from equine fibroblasts using a piggyBac (PB) transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally regulated fashion. The established iPS cell lines express hallmark pluripotency markers, display a stable karyotype even during long-term culture, and readily form complex teratomas containing all three embryonic germ layer derived tissues upon in vivo grafting into immunocompromised mice. Our EiPS cell lines hold the promise to enable the development of a whole new range of stem cell-based regenerative therapies in veterinary medicine, as well as aid the development of preclinical models for human applications. EiPS cell could also potentially be used to revive recently extinct or currently threatened equine species

Small RNA changes en route to distinct cellular states of induced pluripotency

MicroRNAs (miRNAs) are critical to somatic cell reprogramming into induced pluripotent stem cells (iPSCs), however, exactly how miRNA expression changes support the transition to pluripotency requires further investigation. Here we use a murine secondary reprogramming system to sample cellular trajectories towards iPSCs or a novel pluripotent ‘F-class’ state and perform small RNA sequencing. We detect sweeping changes in an early and a late wave, revealing that distinct miRNA milieus characterize alternate states of pluripotency. miRNA isoform expression is common but surprisingly varies little between cell states. Referencing other omic data sets generated in parallel, we find that miRNA expression is changed through transcriptional and post-transcriptional mechanisms. miRNA transcription is commonly regulated by dynamic histone modification, while DNA methylation/demethylation consolidates these changes at multiple loci. Importantly, our results suggest that a novel subset of distinctly expressed miRNAs supports pluripotency in the F-class state, substituting for miRNAs that serve such roles in iPSCs

Bone Marrow Transplantation Results in Human Donor Blood Cells Acquiring and Displaying Mouse Recipient Class I MHC and CD45 Antigens on Their Surface

Background: Mouse models of human disease are invaluable for determining the differentiation ability and functional capacity of stem cells. The best example is bone marrow transplants for studies of hematopoietic stem cells. For organ studies, the interpretation of the data can be difficult as transdifferentiation, cell fusion or surface antigen transfer (trogocytosis) can be misinterpreted as differentiation. These events have not been investigated in hematopoietic stem cell transplant models. Methodology/Principal Findings: In this study we investigated fusion and trogocytosis involving blood cells during bone marrow transplantation using a xenograft model. We report that using a standard SCID repopulating assay almost 100 % of the human donor cells appear as hybrid blood cells containing both mouse and human surface antigens. Conclusion/Significance: Hybrid cells are not the result of cell-cell fusion events but appear to be due to efficient surface antigen transfer, a process referred to as trogocytosis. Antigen transfer appears to be non-random and includes all donor cells regardless of sub-type. We also demonstrate that irradiation preconditioning enhances the frequency of hybrid cell

The Minimal Domain of Adipose Triglyceride Lipase (ATGL) Ranges until Leucine 254 and Can Be Activated and Inhibited by CGI-58 and G0S2, Respectively

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme of lipolysis. ATGL specifically hydrolyzes triacylglycerols (TGs), thereby generating diacylglycerols and free fatty acids. ATGL's enzymatic activity is co-activated by the protein comparative gene identification-58 (CGI-58) and inhibited by the protein G0/G1 switch gene 2 (G0S2). The enzyme is predicted to act through a catalytic dyad (Ser47, Asp166) located within the conserved patatin domain (Ile10-Leu178). Yet, neither an experimentally determined 3D structure nor a model of ATGL is currently available, which would help to understand how CGI-58 and G0S2 modulate ATGL's activity. In this study we determined the minimal active domain of ATGL. This minimal fragment of ATGL could still be activated and inhibited by CGI-58 and G0S2, respectively. Furthermore, we show that this minimal domain is sufficient for protein-protein interaction of ATGL with its regulatory proteins. Based on these data, we generated a 3D homology model for the minimal domain. It strengthens our experimental finding that amino acids between Leu178 and Leu254 are essential for the formation of a stable protein domain related to the patatin fold. Our data provide insights into the structure-function relationship of ATGL and indicate higher structural similarities in the N-terminal halves of mammalian patatin-like phospholipase domain containing proteins, (PNPLA1, -2,- 3 and -5) than originally anticipated

<em>piggyBac</em> transposition reprograms fibroblasts to induced pluripotent stem cells

Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral, lentiviral, adenoviral and plasmid transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies. piggyBac (PB) transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition. Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision, we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies. © 2009 Macmillan Publishers Limited. All rights reserved.Link_to_subscribed_fulltex

The mammalian gene function resource: the International Knockout Mouse Consortium.

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research