Analysis of Cre Activity and Gene Deletion in Conditionally Targeted Mice

Although conditional gene targeting offers tremendous potential for spatially and or temporally specific gene manipulation, the interpretation of the phenotypes may be complicated by unexpected expression of Cre recombinase in the Cre mice. Cre expression may be nonspecific because of position effects or limitations in the selected promoter elements. Not uncommonly, the promoter may also be active at an early stage during mouse development, resulting in extensive deletion in multiple tissues....

Use of Flow Cytometry and Combined DNA Surface Staining for Analysis of Hematopoietic Development in the Xenopus Embryo

Xenopus embryos provide a model for studying the earliest stages in the development of the vertebrate hematopoietic system. This chapter provides detailed procedures describing the production of hematopoietic chimeras in Xenopus embryos and the analysis of these chimeras using flow cytometry. Protocols for analysis include the determination of deoxyribonucleic acid content of hematopoietic cells, staining of cells with antibodies against cell surface antigens and the combined analysis of...

Hemocyte Development During Drosophila Embryogenesis Robert A Schulz and Nancy Fossett

The model genetic organism Drosophila melanogaster has a rudimentary hematopoietic system with two embryonic blood cell types, crystal cells, and plasmatocytes. These distinct lineages provide the animal with an innate immune response and a means to remove apoptotic cells. Genetic analyses of Drosophila hematopoiesis have identified specific genes that function in blood cell formation. Complimentary deoxyribonucleic acid and antibody probes for these hematopoietic factors serve as important...

Quantitative Trait Analysis in the Investigation of Function and Aging of Hematopoietic Stem Cells

Extensive genetically determined quantitative variation exists in the number and function of hematopoietic stem cells in inbred mouse strains. Furthermore, aging of hemato-poietic stem cells is genetically determined. Gene identification of quantitative trait loci involved in the regulation and aging of hematopoietic stem cells would provide novel insights into regulatory mechanisms that are relevant in vivo and may be clinically important. Here we describe strategies for mapping and gene...

New Clonal Assay System for Lymphoid and Myeloid Lineages

Dguo Fetal Thymus Lobe

Hiroshi Kawamoto and Yoshimoto Katsura It has long been unclear how the pluripotent hematopoietic stem cell is restricted to the major lineage progenitors including the progenitors for myeloid, T- and B-cells. This is the result of the absence of a methodology capable of determining the developmental potential of individual progenitors to generate these major lineage cells. We have established such an assay system, termed the multilineage progenitor assay, as a modification of the fetal thymic...

Reconstitution of Hematopoiesis Following Intrauterine Transplantation of Stem Cells

Merchant, Enrico Danzer, and Alan W. Flake In utero hematopoietic stem cell transplantation is an entirely nonmyeloablative approach to achieve mixed hematopoietic chimerism and associated donor-specific tolerance. This chapter provides the rationale and methodologic detail for the administration of stem cells to the preimmune mouse fetus by a variety of routes. The development of murine model systems for in utero transplantation has accelerated progress in the...

Human Mouse Chimeric Organ Culture

For our studies, human umbilical cord blood samples provided by the American Red Cross Cord Blood Program were obtained after informed consent, in conformity with an institutionally approved protocol. Cord blood samples are also available from several commercial sources, for example, AllCells, Berkeley, CA or Cambrex BioScience Walkersville, Walkersville, MD . The thymocyte progenitor population can be transduced with retroviral constructs using VSV-G-pseudotyped retroviruses. 3.9.1. Isolation...

Mouse Embryos or Fetuses

Embryo India Ink Injection Mouse

Anesthesia is performed by intraperitoneal injections. The choice of an anaesthesic is crucial for the success of experiments. Rather than Nembutal or Avertin, used during early stages of gestation, ketamine 0.5-0.7 mL is usually preferred between E12 and E17 for its lower abortion-inducing risk. 3.2.2. Exposing and Preparing the Uterus A vertical incision is performed on one side of the abdominal wall in the anesthetized pregnant mouse see Note 11 . The uterus is exteriorized and the mouse is...

Recombinant Inbred Strains

Recombinant Inbred Strain

In the setting of the analysis of QTL involved in the regulation of HSC, recombinant inbred RI strains offer particular advantages 22,23 . RI strains are commercially available and were generated by repeated inbreeding of F2 mice derived from two parental inbred strains. After 20 generations of inbreeding, a set of RI strains is obtained Fig. 2 . The genome of the individual RI strains of the set is composed of a patchwork of homozygous chromosome segments derived from either progenitor strain,...

Co

Op9 Adipocytes Recovery

Opposite Effect of varying induced levels of HoxB4 gene expression on ES-derived cells co-cultured on OP9. A Images at 40X of OP9 cultures at different doxycycline concentrations. As shown, the density of ES-derived cells small round cells increases dramatically with increases in doxycycline concentration. B Total cell number vs doxycycline concentration. With increasing doxycycline, cell number increases rapidly up to 1 mg mL. C Total cell number of positive cells stained for either...

And Immune Systems of the Avian and Mammalian Embryo

Hematopoiesis Avian

Fran oise Dieterlen-Li vre, Sophie Creuzet, and Josselyne Salaun In vivo experimental approaches that have been designed to study the ontogeny of the hematopoietic system in higher vertebrates are described in the present chapter. The avian embryo is directly available to manipulations in ovo during gastrulation and organogenesis. This permissiveness has led to the design of various approaches that provided crucial insights into the ontogeny of the hematopoietic system, particularly regarding...

Notes

Whole Embryo Culture

When dissecting embryos for whole embryo culture it is important to avoid tearing the placenta or damaging major blood vessels in the yolk sac, as this can compromise oxygen Fig. 1. Schematic representation of tissue graft transplantation under kidney capsule, following exposure of the kidney from the abdominal cavity. A Grasp the kidney capsule with a pair of fine forceps H and use a second pair P to make a small hole in the membrane. Lift the holding forceps H gently to make a space...

Flow Cytometric Analysis of Hematopoietic Development Edward F Srour and Mervin C Yoder

More than 30 yr ago, a collection of cells isolated from the bone marrow were first demonstrated to repopulate hematopoiesis in a radioablated animal. These cells self-renewed while producing all of the blood products and were named hematopoietic stem cells HSCs . Since then, HSCs have been a tremendous boon to both basic science in understanding cell biology and as therapy in a cancer transplant setting. More recent work has shown that the HSCs, and possibly populations of cells residing in...

Injection

Busulfan Administration to Pregnant Dams see Notes 2-4 1. Timed pregnant C57Bl6 J CD45.2 expressing mice are bred to give birth at approx midnight before the day of anticipated transplantation studies. We find that our colony of C57BL6 J mice give birth 19.5 d after coitus and thus plan on performing the transplant on d 20 after coitus. 2. On the morning of d E17 and E18, the pregnant dam is given an intraperitoneal injection of 15.5 mg kg of busulfan see Note 3 . Each dose is delivered...

Ddd

24hpf Zebrafish Drawing Ptu

Nomarski-DIC video-microscopy setup. Continuous lines represent connections through a four-pin Y C cable, dotted lines represent digital Firewire IEEC 1394 connections. Fig. 1. Nomarski-DIC video-microscopy setup. Continuous lines represent connections through a four-pin Y C cable, dotted lines represent digital Firewire IEEC 1394 connections. have used a Panasonic AG6730 but also check Hitachi products . The tape recorder receives the Y C video signal from the camera through a standard...

Retroviral Transduction in Fetal Thymic Organ Culture Bronwyn M Owens Robert G Hawley and Lisa M Spain

Fetal Thymic Organ Culture

T-cell development requires cytokines and intimate contact with stromal cells provided exclusively by the thymus. Consequently, an in vitro model of thymocyte differentiation, fetal thymic organ culture FTOC , has been developed. FTOC recapitulates the normal development of T-cells derived from both mouse and human progenitor populations, providing a more rapid means to study T-cell development compared with alternative in vivo approaches. Furthermore, FTOC is easily amenable to genetic...

Mouse Embryonic Explant Culture System for Analysis of Hematopoietic and Vascular Development

Fine Science Tools Steri 350

In vertebrates, the earliest differentiated cell types hematopoietic and endothelial arise from mesoderm induced during the process of gastrulation. These cells become organized into the blood islands of the extraembryonic yolk sac and are morphologically apparent by around d 7.5 in the mouse. Additional waves of hematopoietic and vasculogenic angio-genic activity later result in the development of definitive hematopoietic lineages and in the formation of the allantois and cardiovascular system...

Knockin Mouse

Knockin Mouse

Expression of introduced sequences may also be achieved by generating a knockin mouse Fig. 1A . In this instance, foreign sequences are introduced into a given locus by homologous recombination. This strategy can be used to express transgenes from known regulatory elements or to modify rather than merely inactivate the targeted gene. Knockin alleles are generally expressed precisely as the endogenous gene where the sequences are introduced. Knockin of a marker gene under the regulatory elements...