In Vivo and In Vitro Assays of Thymic Organogenesis
Julie Gordon, Valerie A. Wilson, Billie A. Moore-Scott, Nancy R. Manley, and C. Clare Blackburn We describe two complementary methods for the study of early thymus organogenesis in the mouse. The first is an in vitro technique for lineage analysis, where a chosen population of cells within the mouse embryo is labeled with a fluorescent cell tracker dye. The embryos are then transferred to whole embryo culture for a defined period, after which time the location of the labeled cells is determined...
Quantitative Trait Analysis in the Investigation of Function and Aging of
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...
A New Clonal Assay System for Lymphoid and Myeloid Lineages
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...
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2.3.6.2. Primitive Erythroid EryP Colony Assays 1. Methylcellulose powder, Fluka cat. no. 64630 see Note 13 . 2. Recombinant human erythropoietin Epogen , 2000 U mL, Amgen cat. no. 3107402. 3. Iscove's modified Dulbecco medium IMDM powder, Gibco-BRL cat. no. 12200-036. 4. Penicillin-streptomycin, 100X 5000 U mL penicillin, 5000 g mL streptomycin , Gibco-BRL cat. no. 15070-063. 5. Fetal bovine platelet poor plasma-derived serum, Animal Technologies Tyler, TX, www.animaltechnologies.com , cat....
Exposure to Fluorescent Light Can Impair Cell Growth and Viability
Because of the known detrimental effects of ultraviolet light on nucleic acids and proteins, it seemed possible that prolonged exposure of cultures to fluorescent light might impair cell growth and viability. To detect and quantify such potential phototoxic effects, we exposed small colonies of mouse erythroleukemia MEL cells for various lengths of time to light passing through four different filters. The impact on cell growth was determined by comparing the cell numbers in the individual...
Wish
WISH is frequently used to evaluate the spatiotemporal expression patterns of hematopoietic genes during ontogeny, and is the basis of our understanding of blood development. Furthermore, WISH is essential for evaluating the effects of perturbing gene expression by overexpression and morpholino-mediated knockdown. More recently, WISH has been implemented as screening criteria to uncover deficiencies in early stem cell specification and definitive hematopoiesis, including the lymphoid and...
Final Purification of HSC by FACS
1. Again all antibody concentrations and incubation times are followed according to the parameters described by the manufacturer guidelines. 2. For our HSC purification we use three different fluorochromes c-Kit conjugated to APC, biotynylated Sca-1 with Streptavidin-PharRed secondary antibody , and the lineage markers B220, CD3, CD4, CD8, CD11B, Flk-1, Gr-1, and TER-119, all directly conjugated to PE. 3. The FACSvantage SE is able to isolate single cells based on the surface antigen bound by...
Reconstitution of Hematopoiesis Following Intrauterine Transplantation of Stem
Elisabeth H. Javazon, Aziz M. 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...
Visualizing Individual Fluorescent Proteins With Different Filters
To compare and quantify the relative signal intensities of the different fluorescent proteins, we exposed individual cells expressing CFP, GFP, YFP, or RFP to light of different wavelengths, using the seven filters, for different lengths of time. As can be seen in Fig. 6, CFP top left can be best visualized by the Cyan GFP filter but also gives a signal with the Endow GFP and the JP1 filter. Thus, the Endow GFP filter Fig. 6. Fluorescent intensities of three GFP variants and of RFP determined...
Expansion and Differentiation of Human Erythroid Progenitors
Several protocols exist for expansion of human erythroid progenitors from cord blood, peripheral blood, or bone marrow. We will describe the current status of our methods to both expand and terminally differentiate human progenitors from cord blood, improving over the published procedures 21,22 with respect to both in vitro life-span of erythroid cells and their ability to undergo terminal differentiation. 3.2.1. Preparation of Mononuclear Cells From Human Umbilical Cord Blood 1. Blood is...
Phenol RedFree Medium and Glass Bottom Vessels Improve Image Quality
The second parameter that contributes to image quality is background fluorescence. To test the influence of phenol red in the culture medium on background fluorescence, we acquired a series of images in media with and without this pH indicator. As shown in Fig. 3, phenol red dramatically increases the background levels, especially when visualizing GFP and RFP with the Endow GFP and TRITC filters, respectively. As a result, the relative signal intensity is decreased, hampering the detection of...
HumanMouse 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...
Verification of Multilineage Reconstitution
1. The recipient animals are given 1 mo to recover and allow the transplanted HSC sufficient time to begin producing the various hematopoietic cell lineages. 2. Determination of engraftment is resolved by peripheral blood sampling and FACS scanning see Note 6 . Sample blood from each animal through a tail vein bleed and collect blood in a tube containing PBS and 5 mM ethylene diamine tetraacetic acid, which inhibits coagulation. 3. Remove red blood cells through a Ficoll purification as...
Info Cjb
Fig. 1. A Flow chart of preparation of cultures from mouse fetal livers and factor combinations used for outgrowth of erythroid and multipotent cells. B Outgrowth of wild type WT, open circles and p53- - black squares erythroid progenitors. Note that after 15 d in culture, wild type cells stop proliferating and die , whereas erythroid cells lacking p53 keep proliferating without limit, leading to cell immortalization. Error bars depict standard deviations of at least four independent...
Materials
1. Green fluorescent protein GFP bone marrow donor mouse male . 2. BL6 syngeneic rescue bone marrow donor mouse female . 3. BL6 bone marrow recipient mouse female . 4. Antibodies list includes antibody and fluorochrome, all from Pharmingen B220 CD45R -PE cat. no. 553090 , CD3-PE cat. no. 553064 , CD4-PE cat. no. 553730 , CD8-PE cat. no. 01045A , CD11B Mac-l -PE cat. no. 553311 , FLK1-PE cat. no. 555308 , Grl Ly6C -PE cat. no. 553128 , TER119 Ly-76 -PE cat. no. 553673 , CD117 c-Kit -APC cat. no....
Temperature Control
The hematovascular system is one of the most temperature sensitive systems of the developing embryo. The heart rate is directly influenced by ambient temperature. Therefore, one of the most important factors in maintaining normal development is accurate environmental temperature control. To keep the temperature constant, we prefer to construct a heater box that surrounds the stage, embryo chamber and microscope optics to ensure stable temperatures and prevent against thermal drift in the optics...
Seeding of a Single Cell Fig 3
3.3.1. Preparation of Wells With a dGuo Lobe 1. Place 200 L of RPMI medium into each well of 96-well V-bottom plate. The medium is supplemented with SCF 10 ng mL , IL-7 5 ng mL , and IL-3 3 ng mL . GM-CSF 1 ng mL can be used instead of IL-3 ref. 22 see Notes 4 and 5 . 2. Place one dGuo-treated FT lobe in each well. 3. Wells of the outer lanes of the plate are usually not used for cultures, since the medium in these wells tend to evaporate more rapidly. 4. In order to prevent evaporation of...
Mouse Embryos or Fetuses
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
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
Fig. 9. 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...
Introduction 1
The mouse hematopoietic system begins to differentiate from the mesodermal germ layer just after E6.5 1,2 . The first site of hematopoiesis, or blood formation, in mammalian embryos is the yolk sac. Clusters of primitive erythroblasts surrounded by endothelial cells emerge from extraembryonic mesoderm as blood islands. The blood islands initially appear in an extraembryonic ring proximal to the embryo proper and then expand to cover the entire yolk sac surface, forming the capillary plexus 3 ....
and Immune Systems of the Avian and Mammalian Embryo
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 Rhv
1. 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...
Info Fpv
vector containing a selectable marker to obtain stable transfectant clones. Cells expressing high levels of murine SCF can either be obtained by producing many subclones and testing these for secretion of SCF by proliferation assays on p53- - erythroblasts for an analogous procedure using avian erythroblasts, see ref. 20 or by sorting the highest expressor clones by flow cytometry using anti CD4 antibodies. In our hands, it is not necessary to purify the SCF from supernatants of secreting...
Flow Cytometric Analysis of Hematopoietic Development Edward F Srour and Mervin
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...
Expansion and Differentiation of Immature Mouse and Human Hematopoietic
Helmut Dolznig, Andrea Kolbus, Cornelia Leberbauer, Uwe Schmidt, Eva-Maria Deiner, Ernst W. M llner, and Hartmut Beug A prerequisite for proper investigation of self-renewal and differentiation of hematopoietic cells is the possibility to obtain large quantities of homogenous primary progenitors under defined conditions, allowing meaningful biochemical and molecular analyses. These cells should show renewal and differentiation characteristics similar to the in vivo situation. The serum-free...
Hematopoiesis Notes
1. Protein localization by immunohistochemistry wash and colorimetric staining of embryos. The black stain is recommended to visualize plasmatocytes. 2. Embryo collection for in situ hybridization. It is advisable to start with large numbers of adult flies 100 to 200 pairs to obtain a sufficient number of embryos to compensate for the potential loss of animals during embryo processing. 3. Embryo collection for protein localization by immunohistochemistry. In general, only 20 to 30 adult pairs...
Conditional Knockin
The Cre-loxP strategy may also be applied for conditional expression of genes in a transgenic or knockin model Fig. 1A . A stopper fragment flanked by loxP Fig. 2. Engineering a conditionally targeted locus. A conditional gene-targeting construct contains a positive selection marker for selection of homologous recombinants flanked by two loxP sites and two homologous arms for directing homologous recombination into wild type locus A third loxP site is placed flanking a critical region of the...
Injection
3.3.1. 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
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. 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
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...
Notes Lhi
1. In hematology, clonal assays have long been used for evaluation of progenitor activity. The CFU-C assay is the most popularly used clonal assay that is effective in determining the developmental potential of individual progenitors towards the erythroid and myeloid lineages 1,2 . In these studies, however, the developmental potential towards the T- or B-cell lineages has not been usually taken into consideration. In contrast, in almost all studies on T- or B-cell development, 103 or more...
Pfhmii
Ascorbic acid 5 mg mL 50 L a-MTG 26 L 2 mL IMDM 30-40 L 1X IMDM Pen-strep to 10 mL total 5 2 mM 25 Mg mL 0.45-0.6 mM 4 U mL 1X 2.3.6.3. Definitive Hematopoietic Colony Assays 1. Methocult , Stem Cells Technologies cat. no. M3434. Contains Epo and other cytokines. 2. 16-gage blunt-end needles, Stem Cells Technologies cat. no. HCC-8110. 3. Petri dishes, 3.5 cm, Becton Dickinson Falcon cat. no. 35-1008. 4. Tissue culture plates, 15 cm, Becton-Dickinson Falcon cat. no. 351013. 5. Preparation of...
Mouse Embryonic Explant Culture System for Analysis of Hematopoietic and
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...
Margaret H Baron md phd
Department of Medicine Hematology-Oncology , Mount Sinai School of Medicine, New York, NY 2005 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Medicine is a trademark of The Humana Press Inc. All papers, comments,...
Conditional Knockin Construct
A conditional knockin construct is generated by introducing a loxP flanked 1.3-kb stopper fragment between the promoter and the modified gene transgene 66 , thus rendering the gene dormant. The stopper fragment causes premature translation termination, and additionally introduces a false translation initiation site and a 5' splice donor site, thereby ensuring that the gene product is not synthesized. Upon Cre expression, the stopper fragment is deleted and the gene is transcribed and...
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...