Constructing a Humanized Mouse Model with Engineered Immunity against HIV

Abstract

Source: Zhen, A., et al. Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model. J. Vis. Exp. (2016).

The video demonstrates a technique to generate a humanized mouse model with stem-cell-based engineered immunity against the human immunodeficiency virus (HIV). Human fetal thymus tissue and engineered hematopoietic stem cells (HSCs) are implanted into the kidney capsule of a severely immunocompromised mouse. The HSCs, which express chimeric antigen receptors (CARs) against HIV, are mixed in a gelatinous protein mixture that promotes their co-localization with thymus tissue. Post-procedure, the HSCs differentiate into various human immune cells, resulting in the development of a humanized mouse model with a functional human immune system against HIV.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Construction of Humanized Mice Engineered with CD4 Chimeric Antigen Receptor

1. Processing Fetal Thymus and Isolating CD34+ HSCs from Fetal Liver
   1. Thymus Processing
      1. Gently wash the thymus in Phosphate-buffered saline (PBS), pH 7.4, in a 15 ml conical tube. Repeat the wash step 3 - 4....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CD34 microbead kit	miltenyi	130-046-702	For sorting human CD34+ progenitor cells
Bambanker	Wako	302-14681	for freezing cells
QIAamp Viral RNA kit	Qiagen	52904	For measuring viral load in the serum
MACSQuant Flow Cytometer	Miltenyi		For flow analysis
BD LSRFortessa™	BD biosciences		For flow analysis
Hyaluronidase	Sigma	H6254-500MG	For tissue digestion
Deoxyribonuclease I	Worthington	LS002006	for tissue digestion
Collagenase	Life technology	17104-019	for tissue digestion
CFX Real time PCR detection system	Biorad		For measuring viral load and gene expression
Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ	The Jackson Laboratory	5557	For constructing the humanized mice
Penicillin Streptomycin (Pen Strep)	Thermo Fisher Scientific	10378016	For culturing cells
piperacillin/tazobactam	Pfizer	Zosyn	Anti-fungal
Amphotericin B (Fungizone antimycotic)	Thermo Fisher Scientific	15290-018	Anti-fungal
AUTOCLIP Wound Clips, 9 mm - 1000 units	Becton Dickinson	427631	For surgery
Sterile Poly-Reinforced Aurora Surgical Gowns, 30 per case	Medline	DYNJP2707	For surgery
Sutures, 4-0, vicryl	Owens and Minor	23000J304H	For surgery
Alcohol prep pads	Owens and Minor	3583006818	For surgery
Gloves, surgical, 6 1/2	Owens and Minor	4075711102	For surgery
Yssel's Serum-Free T-Cell Medium	Gemini Bio-products	400-102	For CD34+ cell transduction
Human Serum Albumin	Sigma-Aldrich	A9511	For CD34+ cell transduction