Source: Dagur, R. S. et al., Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis. J. Vis. Exp. (2019)
This video demonstrates a method for generating a dual humanized mouse model with a human immune system and liver cells. It involves transplanting human liver cells and hematopoietic stem and progenitor cells into a transgenic immunodeficient mouse via intrasplenic injection, leading to engraftment of liver cells and development of human immune cells.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Processing of Umbilical Cord Blood and the Isolation of Human Hematopoietic Stem/Progenitor Cells (HSPCs)
2. Preparation of Human Hepatocytes (HEPs) for Transplantation
3. Animal Handling, Screening, Genotyping, and Treatment for Human HSPC and Hepatocyte Transplantation
Figure 1: Schematic of the enrichment of CD34+ cells from cord blood. (A) Cord blood is layered on lymphocytes separation medium (LSM) and centrifuged to isolate the buffy coat. (B) LS columns are placed on a magnetic stand and rinsed with a BSA buffer, followed by adding a buffy coat. Cells positive for CD34 are trapped in the columns, and CD34-cells are eluted in separate tubes. Trapped CD34+ cells in column resins are plunged with a plunger, and the cells are collected in a new tube.
Figure 2: Schematic view of the experimental design for the dual reconstitution of humanized liver and immune system mice, followed by HIV-1 infection. TK-NOG mice are injected with ganciclovir (GCV) at a dose of 6 mg/kg, 2x a day, on day -7 and day -5, followed by a treosulfan injection on days -3, -2 and -1. To screen the mice for transplantation (Tx), an alanine aminotransferase (ALT) assay is performed one day before the surgery, and mice with ALT levels of >200 and <600 U/L are selected. After transplantation, the mice are checked for a reconstitution of the human immune system by flow cytometry (FACS) and for liver reconstitution by assessing their albumin level using ELISA. The mice are infected with HIV-1 5 weeks before they are sacrificed.
The authors have nothing to disclose.
27G1/2" needles | BD biosciences | 305109 | |
30G1/2" needles | BD biosciences | 305106 | |
5 mL polystyrene round-bottom tube 12 x 75 mm style | Corning | 352054 | |
BD 1 mL Tuberculin Syringe Without Needle | BD biosciences | 309659 | |
BD FACS array bioanalyzer | BD Biosciences | For purity check of eluted CD34+ cells | |
BD FACS array software | BD Biosciences | Software to analysis acquired CD34+ cell on FACS array | |
BD FACS lysing solution | BD Biosciences | 349202 | To lyse red blood cells |
BD LSR II | BD Biosciences | Instrument for acquisiton of flow cytometry samples | |
BD Vacutainer Plastic Blood Collection Tube | BD biosciences | BD 367874 | To collect Cord blood |
Bovine Serum Albumin | Sigma-aldrich | A9576 | |
CD34 MicroBead Kit, human | Miltenyi Biotec | 130-046-702 | For isoation of CD34+ HSPC |
CD34-PE, human | Miltenyi Biotec | 130-081-002 | Antibody used for purity check of eluted CD34+ cells |
Cell counting slides | Bio-rad | 1450015 | |
ChargeSwitch gDNA Mini Tissue Kit | Thermofisher scientific | CS11204 | For extraction of genomic DNA from ear piece |
Cotton-tipped applicators | McKesson | 24-106-2S | |
DMSO (Dimethyl sulfoxide) | Sigma-aldrich | D2650-5X5ML | |
Extension set Microbore Slide Clamp(s) Fixed Male Luer Lock. L: 60 in L: 152 cm PV: 0.55 mL Fluid Path Sterile | BD biosciences | 30914 | Attached to dispensing pippet and to load with HSPC and HEP suspesion |
FACS Diva version 6 | BD Biosciences | Flow cytometer software required for acqusition of sample | |
Fetal Bovine Serum (FBS) | Gibco | 10438026 | |
FLOWJO analysis software v10.2 | FLOWJO, LLC | Flow cytometry analysis software | |
Ganciclovir | APP Pharmaceuticals, Inc. | 315110 | Prescripition drug |
Greiner MiniCollect EDTA Tubes | Greiner bio-one | 450475 | |
Hepatocytes thawing medium | Triangle Research Labs | MCHT50 | |
Horizon Open Ligating Clip Appliers | Teleflex | 537061 | To hold the ligating clips |
Human hepatocyte | Triangle Research Labs | HUCP1 | Cryopreserved human hepatocytes, induction qualified |
Iris Scissors, Straight | Ted Pella, Inc. | 13295 | |
Lancet | MEDIpoint | Goldenrod 5 mm | |
LS columns | Miltenyi Biotec | 130-042-401 | Used to entrap CD34+ microbeads (positive selection) |
Lymphocyte Separation Medium (LSM) | MP Biomedicals | 50494 | For isoation of lymphocytes from peripheral blood |
MACS MultiStand | Miltenyi Biotec | 130-042-303 | Holds Qudro MACS seperator and LS columns |
McPherson-Vannas Micro Dissecting Spring Scissors | Roboz Surgical Instrument Co. | RS-5605 | Used to make an incision on skin to expose spleen |
Micro Dissecting Forceps | Roboz Surgical Instrument Co. | RS-5157 | To hold and pull out spleen from peritoneal cavity |
Mouse CD45-FITC | BD Biosciences | 553080 | Mouse-specific |
PBS (Phosphate Buffered Saline) | Hyclone | SH30256.02 | |
Qudro MACS separator | Miltenyi Biotec | 130-090-976 | holds four LS columns |
RPMI 1640 medium | Gibco | 11875093 | |
StepOne Plus Real Time PCR | Applied Biosystems | Instrument used to genotype | |
Stepper Series Repetitive Dispensing Pipette 1ml | DYMAX CORP | T15469 | Used to dispense HSPC and HEP supension in controlled manner |
TaqMan Gene Expression Master Mix | Thermofisher scientific | 4369016 | |
TC20 automated cell counter | Bio-rad | 1450102 | |
TK-NOG mice | Provided by the Central Institute for Experimental Animals (CIEA, Japan; Drs. Mamoru Ito and Hiroshi Suemizu) | ||
Treosulfan | Medac GmbH | Provided by Dr. Joachim Baumgart (medac GmbH) | |
Trypan Blue | Bio-rad | 1450022 | |
Vannas-type Micro Scissors, Straight, 80mm L | Ted Pella, Inc. | 1346 | Used to make an incision on skin to expose spleen |
Weck hemoclip traditional titanium ligating clips | Esutures | 523700 | To ligate the spleen post-injection |
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