Immune-privileged tissues formed from immunologically cloaked mouse embryonic stem cells survive long term in allogeneic hosts.

The immunogenicity of transplanted allogeneic cells and tissues is a major hurdle to the advancement of cell therapies. Here we show that the overexpression of eight immunomodulatory transgenes (Pdl1, Cd200, Cd47, H2-M3, Fasl, Serpinb9, Ccl21 and Mfge8) in mouse embryonic stem cells (mESCs) is sufficient to immunologically 'cloak' the cells as well as tissues derived from them, allowing their survival for months in outbred and allogeneic inbred recipients. Overexpression of the human orthologues of these genes in human ESCs abolished the activation of allogeneic human peripheral blood mononuclear cells and their inflammatory responses.

Observation of Fluorescent Proteins in Fixed Cells.

Fluorescent proteins (FPs) are popular reporters available for gene expression detection and determination of cellular identities in the mouse. This protocol can be used to detect green fluorescent protein spectral variants and proteins labeled with the fusion tag dsRed in fixed cells.

Observation of Fluorescent Proteins in Living Cells.

Frequently, there is a need for fluorescent protein detection in mouse cell cultures, including embryonic stem cells or their differentiated derivatives, primary and transformed cells. Here, cells expressing green fluorescent protein-labeled proteins are observed using fluorescent microscopy.

Preparation of Polymerase Chain Reaction Template DNA from Mouse Tail Tissue.

A simple cell or tissue lysate can provide a sufficient quality and amount of template DNA for polymerase chain reaction (PCR). In this protocol, a small piece from the tip of the tail is removed and processed using hot sodium hydroxide and Tris (HotSHOT).

Isolation of High-Molecular-Weight DNA from Mouse Tail Tips.

DNA samples are prepared from mouse tail tips by Proteinase K digestion and subsequently extracted. The resulting preparation is suitable for use in Southern blotting or polymerase chain reaction (PCR).

Isolation of High-Molecular-Weight DNA from Mouse Yolk Sacs and the Like.

Often, genotyping of mouse embryos is required, and a small part, such as the yolk sac, can be used for this purpose. Here, DNA samples are prepared from extra-embryonic tissues by digestion with Proteinase K and subsequent extraction. The yolk sac of mid-gestation or later-stage embryos provides a sufficient amount of DNA for Southern analysis.

In Vitro Screen to Identify Silent but Activatable (S/A) Integration Sites for a Tetracycline-Inducible Transgene in Mice.

To obtain ubiquitous or simply widespread transgene expression from a single stable integrant transgene is quite challenging because the random genomic integration sites of transgenes may create expression variation or frequent silencing. The tetracycline (Tet)-inducible system requires two reliable working transgenes, one for the tetracycline transactivators (tTA or rtTA) and one for the responder transgene driven by the promoter. Therefore, the challenge of getting this system working properly is a serious prospect.

Subcutaneous Injection of Pluripotent Stem Cells in Mice.

This protocol describes the production of teratomas and teratocarcinomas used to determine the in vivo developmental potential of adult or embryonic tissues, including early-stage embryos, somites, and tail bud, as well as embryonic and other pluripotent stem cells. Resulting tumors are screened by histology and markers to assess differentiation of the tissues. Methods and sites may vary, depending on cell and tissue type.

Visualization of Fluorescent Protein Expression in Whole-Mount Postimplantation-Stage Mouse Embryos.

Fluorescent protein (FP)-expressing transgenic mice are powerful genetic resources for marking both live and fixed cells and tissues. Expression in live embryos requires only dissection and visualization using an appropriate microscope. Fixation can compromise FP activity.

Administration of Gonadotropins for Superovulation in Mice.

For experiments that require large numbers of preimplantation mouse embryos, such as microinjection of zygotes, gonadotropins are administered to females before mating to increase the number of oocytes that are ovulated (i.e., to induce superovulation).

Preparation of DNA from Embryonic Stem Cells or Other Cultured Cells.

Characterization of embryonic stem (ES) cell-mediated genome alterations, including random insertional transgenesis, gene trapping, gene targeting, and site-specific recombinase-mediated changes, is performed mostly at the ES cell level, before the introduction of these alterations into a mouse. A detailed characterization requires a larger amount of DNA than is required for the initial detection of the candidates for the desired alteration. This protocol describes the preparation of DNA from a 10-cm tissue culture plate.

Testing Serum Batches for Mouse Embryonic Stem Cell Culture.

The variability in embryonic stem (ES) cell culture is due primarily to serum. Serum is typically produced in large batches from many animals. However, samples may differ depending on the age and diet of the animals, the country of origin, and other factors creating lot-to-lot variations.

Reprogramming Mouse Fibroblasts with Transposons.

In 2006, Shinya Yamanaka and his student Kazutoshi Takahashi showed that the expression of only four specific genes is sufficient to reprogram fully differentiated somatic cells into pluripotent stem cells. These cells, termed induced pluripotent stem (iPS) cells, share many of their characteristics with embryonic stem (ES) cells. In this protocol, we describe one of the simplest ways of generating iPS cells from mouse fibroblasts.

Integrating Transposon Transgenes into Mouse Fibroblasts Using Chemical Methods.

Mouse embryonic fibroblasts can be reprogrammed to ES cell-like pluripotent stem cells by the forced expression of four transcription factors-OCT4, SOX2, KLF4, and c-MYC. The transposon system has proven effective as a vehicle for the delivery of transgenes into fibroblasts and for successful reprogramming to induced pluripotent stem (iPS) cells. We found that FuGENE HD transfection reagent can be effective for mouse embryonic fibroblasts (MEFs) to generate induced pluripotent stem cells (iPSCs) with the transposon transgenes.

Integrating Transposon Transgenes into Mouse Fibroblasts by Electroporation.

Mouse embryonic fibroblasts can be reprogrammed to embryonic stem (ES) cell-like pluripotent stem cells by the forced expression of four transcription factors-OCT4, SOX2, KLF4, and c-MYC. The transposon system has proven effective as a vehicle for the delivery of transgenes into fibroblasts and for successful reprogramming to induced pluripotent stem (iPS) cells. This protocol is designed for use with the Neon electroporation system.

Shipment of Live Preimplantation-Stage Mouse Embryos.

Sharing genetically modified mouse models is a very important part of collaboration between researchers. Shipping live animals around the world is inconvenient, expensive, and cumbersome because of the variety of international regulations and paperwork. The issue of health status differences between animal facilities is of great importance; traditionally, imported animals are quarantined to determine their health status and avoid the introduction of undesirable pathogens.

Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies by Hanging-Drop Cultures.

Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. There are an increasing number of differentiating culture conditions that can bias the differentiation of ES cells into desired cell types.

Selecting Female Mice in Estrus and Checking Plugs.

The female mouse estrous cycle is divided into four phases: proestrus (development of ovarian follicles), estrus (ovulation), metestrus (formation of corpora lutea), and diestrus (beginning of follicle development for next ovulation and elimination of previous oocytes). The appearance of the epithelium of the external genitalia is used to identify the stage of the estrous cycle of a female mouse. This is usually easier to see in strains with either no or only light skin pigmentation.