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Differential Screens with Subtracted PCR-Generated cDNA Libraries from Subregions of Single Mouse Embryos

Differential gene expression is an essential mechanism for the development of a single-celled zygote into a patterned multicellular organism with differentiated cell types and tissues. Therefore, the identification of differentially expressed genes provides an important ...

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Analysis of mRNA Expression During Preimplantation Development

Preimplantation-stage mammalian embryos are very simple morphologically, being composed of mostly round cells that exhibit only a limited range of changes in cell shape, namely those associated with compaction and formation of the trophectoderm cell lineage. This morphological ...

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HPLC-Based mRNA Differential Display

Analysis of the genetic and molecular mechanisms of embryogenesis requires effective methods to find and isolate genes that are differentially expressed in normal and altered conditions. Comparative genomic screens can lead to the discovery of known or novel transcripts that chan ...

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Production of Transgenic Drosophila

The generation of transgenic Drosophila has become a standard technique for the investigation of a wide variety of biological questions and phenomena. Virtually any cloned gene can be inserted into the genome using the P transposable element system. The gene of interest is first subcloned b ...

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Transgenic Manipulation of the Sea Urchin Embryo

The cis-regulatory regions of developmentally active genes offer an accessible inroad from which to approach an understanding of the transcriptional networks that coordinate embryogenesis (1). In a developmental context, cis-regulatory regions control not only transcrip ...

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Transgenic Zebrafish

The zebrafish has become a popular model system for genetic studies of vertebrate development. Zebrafish are amenable to such studies because the generation time of zebrafish is short, adults are small and easy to maintain, and females typically lay up to several hundred eggs per mating. Early d ...

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Incorporation of Genetically Modified Cells in Chicken Chimeras

The production and study of transgenic mice has proven invaluable in dissecting the complex genetic regulatory events that control mammalian development. Although the chick embryo is a classic model system in developmental biology, its scope is limited by the inability to routinely and ...

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Production of Avian Chimeras and Germline Transmission

Fertilization in chickens occurs in the infundibulum of the reproductive tract within 15 min after ovulation of the yolk. The first cleavage division occurs 5 h later as the egg enters the shell gland. As the shell is calcified during the next 20-22 h, the embryo develops into a radially symmetric dome of c ...

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Long-Term Culture of Chicken Blastodermal Cells (CBCs) and Selection of Transfected CBCs Using Antibiotic Resistance

Chicken blastodermal cell (CBC) culture is a useful tool for the production of germline and somatic chimeras. The long-term culture of CBCs may be used for studying the in vitro effects of various cytokines and growth factors at different stages of embryonic development (1). In addition, cell cul ...

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Nuclear Transplantation and Cloning in Mammals

Nuclear transplantation has been used for many years as an investigative tool with which to study the interactions between the nucleus and cytoplasm in early embryos and with which to characterize the developmental potency of embryonic and adult cell nuclei. As reviewed in a recent book on the s ...

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Production of Transgenic Mice

The term “transgenic ” has been used since the beginning of the 1980s to define animals that had incorporated foreign DNA into their genome (1 ,2).Currently, transgenic animals include various species such as fish,poultry,rats,rabbits,pigs, sheep,goats,cattle,and mice (3 ,4).Mice r ...

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Production of Transgenic Mice with Yeast Artificial Chromosomes

The stable introduction of recombinant DNA into the germline of mammals is one of the major technological advances in biology and has greatly facilitated the study of gene function in development and disease,as well as the development of biotherapeutics. Landmark experiments conduct ...

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Gene-Targeting Strategies

Gene targeting in mouse embryonic stem (ES) cells has become a routine methodology to study gene function in vivo (1). Indeed, gene-targeting core facilities have been established at numerous institutions to facilitate the generation of targeted ES cell lines and the production of mouse ch ...

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Chimeric Animals and GermlineTransmission

Gene targeting by homologous recombination in pleuripotent embryonic stem (ES) cells provides a powerful tool to introduce specific mutations in the genome of intact animal (1-5). It, therefore, allows to unravel the function of genes that control development and differentiation in the ...

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Conditional Gene Knockout Using Cre Recombinase

The directed introduction of null mutations into defined genes has proven invaluable in elucidating gene function in a variety of experimental organisms. In the last decade or so this approach has been extended to mice (1) by the combined use of homologous recombination in murine embryonic st ...

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The Expanding Tool Kit for Hematopoietic Stem Cell Research

Hematopoietic stem cells (HSC) play critical roles in maintaining blood cell production for the lifetime of the organism. Considerable progress has been made in their isolation from mouse bone marrow to high levels of purity based on a combination of cell-surface phenotype and functional ...

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Isolation of Quiescent Murine Hematopoietic Stem Cells by Homing Properties

A major challenge facing investigators working in the field of hematopoietic stem cell (HSC) biology has been to develop a strategy to purify rare primitive HSCs from bone marrow. Several methods have been available including the commonly used technique of isolating HSCs based on a specific c ...

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Mobilization of Hematopoietic Stem and Progenitor Cells in Mice

Animal models have added significantly to our understanding of the mechanism(s) of hematopoietic stem and progenitor cell (HSPC) mobilization. Such models suggest that changes in the interaction between the HSPC and the hematopoietic microenvironmental ‘niche’ (cellular and e ...

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Transmigration of Human CD34+ Cells

Understanding mechanisms responsible for engraftment of hematopoietic stem cells (HSC) is important to achieve successful HSC transplantation. Homing of HSC to the bone marrow niche is believed to be a crucial step for engraftment. However, the molecular mechanisms that regulate HSC ...

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Flow Cytometry-Based Cell Cycle Measurement of Mouse Hematopoietic Stem and Progenitor Cells

The balance between the quiescent hematopoietic stem cell (HSC) and the highly proliferative hematopoietic progenitor compartments maintains homeostasis in the hematopoietic system. Therefore, the entry of HSCs into the cell cycle and the rate of proliferation of hematopoiet ...

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