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Authors: Els Verhoeyen and Francois-Loic Cosset

Adapted from Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). CSHL Press, Cold Spring Harbor, NY, USA, 2007.

INTRODUCTION

In the protocol presented here, hematopoietic stem cells (HSCs) are specifically transduced with a vector displaying the HSC-activating polypeptides, stem cell factor (SCF) and thrombopoietin (TPO). Targeted HSC transduction is evaluated in the non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model.

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Authors: Scott R. Hutton and Larysa H. Pevny

Corresponding author: [email protected]

INTRODUCTION

The ability to prospectively identify and characterize neural progenitor cells in vivo has been difficult due to a lack of cell-surface markers specific for these cell types. A widely used in vitro culture method, known as the Neurosphere Assay (NSA), has provided a means to retrospectively identify neural progenitor cells as well as to determine both their self-renewal capacity and their ability to generate the three primary cell types of the nervous system: neurons, astrocytes, and oligodendrocytes. Today, combined with the establishment of multiple transgenic mouse strains expressing fluorescent markers and advances in cell isolation techniques such as fluorescence-activated cell sorting (FACS), the NSA provides a powerful system to prospectively elucidate neural progenitor characteristics and functions. Here we describe methods for the isolation, culture, and dif

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Authors: Sohyun L. McElroy and Renee A. Reijo Pera

Corresponding author: [email protected]

INTRODUCTION

In somatic cell nuclear transfer (SCNT), the nucleus of a somatic cell is transferred to an enucleated oocyte for reprogramming to an embryonic cell state through the use of the endogenous machinery. SCNT technology has been used to produce offspring, establish embryonic stem cells, and study epigenetic reprogramming, as mediated by oocytes, in several animal species. In humans, there are ethical and practical issues that limit availability of oocytes donated by women of reproductive age specifically for research. Thus, there is a need to more exhaustively explore alternatives, including oocyte sources and different SCNT protocols. Nuclear transfer (NT) techniques are important factors that impact development of NT embryos. The procedures of enucleation of oocyte genetic material and introduction of the donor nucleus vary depending on species and laboratories. Hoechst st

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Authors: Roxanne Holmes and Juan Carlos Zúñiga-Pflücker

Corresponding author: [email protected]

INTRODUCTION

Differentiation of mouse embryonic stem cells (ESCs) or hematopoietic stem cells (HSCs) from fetal liver or bone marrow into T-lymphocytes can be achieved in vitro with the support of OP9-DL1 cells, a bone-marrow-derived stromal cell line that ectopically expresses the Notch ligand, Delta-like 1 (Dll1). This approach provides a simple, versatile, and efficient culture system that allows for the commitment, differentiation, and proliferation of T-lineage cells from different sources of stem cells. This article contains a series of protocols, the first of which describes the establishment, maintenance, and storage of OP9 and OP9-DL1 cells. Subsequent protocols detail how to co-culture the OP9 and OP9-DL1 cells with either ESCs or HSCs from fetal liver or bone marrow, leading to in vitro differentiation of the stem cells into lymphocytes.

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Method:

1. Grow cells on sterile coverglasses or chamber slides overnight.

• Rinse briefly with PBS
• Fix cells by incubation with one of the following methods:
  • 1% formalin in PBS for 10 minutes
  • 80% methanol in PBS for 10 minutes
  • Cold acetone for 5 minutes, air dry
• 4% paraformahyde

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Coat the Plate:

1. Dilute Low-Endotoxin/Azide-Free sterile unlabeled capture antibody (BioLegend’s LEAF™ format antibodies are specifically designed for this assay) to a final concentration of 0.5–4 μg/ml in sterile Coating Buffer and transfer 100 μl/well to a high affinity binding PVDF membrane ELISPOT plate (e.g., Millipore; Cat. No. MAIPS-4510).

• Store plates overnight in humidified box at 4°C or at 37°C for ≥ 4 hours in humidified atmosphere.

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Methods:

1. Deparaffinize and rehydrate sections:

• 3 x 3´ Xylene
• 3 x 2´ 100% Ethanol
• 1 x 2´ 95%, 80%, 70% Ethanol (each)
• 1 x 5´ 1X PBS
• Antigen retrieval methods:

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Solutions

1. Solution A

• 5mM potassium ferricyanide, crystalline
• 5mM potassium ferrocyanide, trihydrate
• X-gal Stock Solution
  • 40mg/mL in DMSO (100mg in 2.5ml DMSO)
• Final X-gal Solution

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I. BrdU Treatment

Mice were injected i.p. with 0.01 mL/g bromodeoxyuridine (Amersham Cat.# RPN 201). Animals were sacrificed two hours later and perfusion fixed with 4% paraformaldehyde/PBS. Mammary glands were removed, post fixed in 4% paraformaldehyde overnight then paraffin embedded and sectioned and mounted onto slides. Alternatively, mice were sacrificed and the glands removed immediately without fixation and frozen for cryostat sectioning. Not surprisingly, the morphology is best on the paraffin sections. (Attached photomicrograph shows a labeled duct in the abdominal gland from a post-pubertal mouse.)

II. BrdU Detection

A. Perfusion fixed, paraffin sections:

1. Deparaffinize sections using standard xylene/alcohol procedures.

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Materials Needed

1. PAP pen (Abcam ab2601)

• Histochoice MB Fixative (Electron Microscopy Sciences 64115-01)
• 10xPBS (Fisher BP3994)
• Triton X (Roche 12754423)
• Bovine serum albumin (Sigma A8022-500G)
• Mouse CD31 [PECAM] (BD Biosciences 555025)
• Rabbit alpha smooth muscle actin (Abcam ab5694)