S6K1 controls pancreatic ß cell size independently of intrauterine growth restriction.

Type 2 diabetes mellitus (T2DM) is a worldwide heath problem that is characterized by insulin resistance and the eventual loss of ß cell function. As recent studies have shown that loss of ribosomal protein (RP) S6 kinase 1 (S6K1) increases systemic insulin sensitivity, S6K1 inhibitors are being pursued as potential agents for improving insulin resistance. Here we found that S6K1 deficiency in mice also leads to decreased ß cell growth, intrauterine growth restriction (IUGR), and impaired placental development. IUGR is a common complication of human pregnancy that limits the supply of oxygen and nutrients to the developing fetus, leading to diminished embryonic ß cell growth and the onset of T2DM later in life. However, restoration of placental development and the rescue of IUGR by tetraploid embryo complementation did not restore ß cell size or insulin levels in S6K1-/- embryos, suggesting that loss of S6K1 leads to an intrinsic ß cell lesion. Consistent with this hypothesis, reexpression of S6K1 in ß cells of S6K1-/- mice restored embryonic ß cell size, insulin levels, glucose tolerance, and RPS6 phosphorylation, without rescuing IUGR. Together, these data suggest that a nutrient-mediated reduction in intrinsic ß cell S6K1 signaling, rather than IUGR, during fetal development may underlie reduced ß cell growth and eventual development of T2DM later in life.

Thawing embryonic stem (ES) cells from a 96-well plate.

INTRODUCTION: The most widely used method to alter the genome of embryonic stem (ES) cells is to introduce a specifically designed DNA fragment using electroporation. The DNA will then integrate into the genome of ES cells. Colonies of cells containing the exogenous DNA are then picked, expanded, replica-plated, frozen in 96-well plates, and used as a source for genomic DNA preparation for genotyping. After ES candidate clones are identified by genomic Southern blot or polymerase chain reaction (PCR), the method of rescue of the cells from the frozen 96-well plates is very important. This protocol describes a method for thawing such cells.

Aggregation chimeras: combining ES cells, diploid, and tetraploid embryos.

During the past 40 years, mouse chimeras have served as invaluable tools for studying not only genetics but also embryonic development, and the path from undifferentiated cell populations to fully committed functional cell types. This chapter gives a description of the early events of cell commitment and differentiation in the pre-and postimplantation-stage embryo. Next, a discussion follows highlighting the most commonly used as well as more recently developed applications of various cell types and origins used in the production of chimeras. Finally, detailed protocols and trouble-shooting suggestions will be presented for each of the steps involved.

Bacterial artificial chromosome transgenesis through pronuclear injection of fertilized mouse oocytes.

In the mouse, conventional transgenes often produced unpredictable results mainly because they were too small to recapitulate a natural gene context. Bacterial artificial chromosomes (BACs) are large enough to encompass the natural context of most mammalian genes and consequently deliver more reliable recapitulations of their endogenous counterparts. Furthermore, recombineering methods now make it easy to engineer precise changes in a BAC transgene. Consequently, BACs have become the preferred vehicle for mouse transgenesis. Here, we detail methods for BAC transgenesis through pronuclear injection of fertilized oocytes.

Immunohistochemistry of embryo sections.

INTRODUCTIONThis protocol describes how to localize an antigen in cells and tissues using embryo sections attached to glass slides. The method outlined here uses alkaline-phosphatase-coupled secondary antibody; horseradish-peroxidase-coupled secondary antibody can be used as an alternative.

Karyotyping mouse cells.

INTRODUCTIONThe majority of mouse chromosome preparations for banding are now made by air-drying and, in essence, require the production of a cell suspension as a starting point. Some samples such as blood cultures, ascitic fluids, or cells growing in suspension will already be in suspension; others, such as bone marrow, solid tumors, or cells growing as attached layers in culture must be converted to suspensions. The basic steps in karyotyping and banding embryonal carcinoma cells are outlined below.

Analyzing glucose phosphate isomerase isozymes in chimeric mouse tissues by electrophoresis.

INTRODUCTIONMouse strains carry different alleles at the ubiquitously expressed Gpi1 (glucose phosphate isomerase) locus (Gpi1(a), Gpi1(b), Gpi1(c)), and this is the basis for a widely used method for determining the genotypic composition of different tissues in mouse chimeras. To determine chimerism by this method, it is necessary to separate the differently charged isozymes from tissue homogenates electrophoretically and to visualize them using a color reaction. Because GPI is a dimer, tissues that normally form by cell fusion (e.g., skeletal muscle) have a heterodimeric form of GPI in a chimera.

The 7th Transgenic Technology meeting: debut for "down under" (http://www.tasq.uq.edu.au/TT2007).

The 7th Transgenic Technology meeting was held in Brisbane, Australia on February 12-14, 2007. Not only did this gathering mark a milestone as it was hosted outside the European continent for the first time, but also because it was the initial meeting to be held on behalf of the new International Society for Transgenic Technologies (ISTT, http://www.transtechsociety.org/ ). As in previous years, the topics were aimed towards both a scientific as well as a technical audience. The subjects covered a wide range of cutting edge applications in the field of genetic modifications in animal models, with the focus on (but by no means limited to) mice. True to the meetings tradition, a large emphasis was also laid on discussions about the management of transgenic production units. With the beautiful Australian sun shining over the venue, and a large number of exceptional speakers, this was a most pleasant and informative conference.

Developmental and adult phenotyping directly from mutant embryonic stem cells.

Tetraploid embryo complementation assay has shown that mouse ES cells alone are capable of supporting embryonic development and adult life of mice. Newly established F(1) hybrid ES cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell-based genetics feasible. Here we report the establishment and characterization of 12 new F(1) hybrid ES cell lines and the use of one of the best (G4) in a gain- and loss-of-function genetic study, where the in vivo phenotypes were assessed directly from ES cell-derived embryos. We found the generation of G4 ES cell-derived animals to be very efficient. Furthermore, even after two consecutive rounds of genetic modifications, the majority of transgenic lines retained the original potential of the parental lines; with 10-40% of chimeras producing ES cell-derived animals/embryos. Using these genetically altered ES cells, this success rate, in most cases, permitted the derivation of a sufficient number of mutants for initial phenotypic analyses only a few weeks after the establishment of the cell lines. Although the experimental design has to take into account a moderate level of uncontrolled damage on ES cell lines, our proof-of-principle experiment provides useful data to assist future designs harnessing the power of this technology to accelerate our understanding of gene function.

Fixation of mouse embryos and tissues.

INTRODUCTIONThis protocol provides methods for fixation of mouse embryos and tissues with paraformaldehyde, Bouin's fixative, or methanol/DMSO solution.

Sectioning mouse embryos.

INTRODUCTIONThis protocol provides methods and tips for sectioning mouse embryos and transferring the sections to a microscope slide.

Handling mouse blastocysts for fixation.

INTRODUCTIONWhole blastocysts or sections of blastocysts can be fixed and used for histological studies, including in situ hybridization and immunohistochemistry. As described in this protocol, different handling techniques are required for whole versus sectioned embryos. For sectioning, it is more convenient to transfer blastocysts into the ampulla of an oviduct prior to fixation. The oviduct serves as a carrier that is easy to handle during subsequent processing for histology.

Embedding mouse embryos and tissues in wax.

INTRODUCTIONThis protocol describes how to embed mouse tissues and embryos (large and small) in wax. The specimens must be dehydrated prior to embedding. Embedded samples are subsequently sectioned and used for in situ hybridization and immunohistochemistry experiments.

Preparing glass slides and coverslips for in situ hybridization.

INTRODUCTIONPrecleaned glass slides are of high enough quality for both in situ and immunohistochemical techniques. However, for in situ hybridization, the slides need to be treated with diethyl pyrocarbonate (DEPC) so that any RNase attached to them is destroyed. The slides also need to be coated with 3-triethoxysilylpropylamine (TESPA) or poly-L-lysine so that the sections adhere tightly and do not detach during subsequent extensive washing procedures. This protocol describes techniques for coating slides with TESPA and poly-L-lysine. There are advantages and disadvantages to each coating method. TESPA-treated slides can be stored for a long time, but the sections do not adhere tightly until after drying. Poly-L-lysine-coated slides need to be made fresh, but the sections adhere immediately on contact with the surface. This protocol also describes how to prepare coverslips for in situ hybrization by coating them in a siliconizing solution.

Dewaxing and Rehydrating Sections prior to In Situ Hybridization.

INTRODUCTIONThis protocol describes how to remove wax from embryo or tissue sections that have been affixed to glass slides. Traditionally, xylene has been used for this purpose, but less toxic solutions can also be employed. The embryo or tissue sections are then progressively rehydrated for compatibility with subsequent alcohol stains, aqueous stains, immunohistochemistry, or in situ hybridization.

In situ hybridization of mouse embryo and tissue sections with radiolabeled RNA probes.

INTRODUCTIONThis protocol describes in situ hybridization of embryo and tissue sections with (35)S-labeled, single-stranded, antisense RNA probes (riboprobes). Protocols have also been developed for in situ hybridization to tissue sections using nonradiolabeled RNA probes that can be detected with antibodies coupled to alkaline phosphatase and a chromogenic substrate. Nonradioactive methods have the advantage that the results can be obtained relatively quickly, but the sensitivity is probably lower than with radioactive probes. In addition, care must be taken to optimize the amount of each probe used in the hybridization reaction.