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....

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...

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...

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...

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...

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...