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.
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
The authors have nothing to disclose.
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 |
.