Source: Zhang, G., et al. Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model. J. Vis. Exp. (2017)
This video describes the protocol for intracardiac injection of retroviruses into the transgenic mouse to ensure gene transfer into proliferating pancreatic β premalignant lesions. This method can also help in assessing the functions of candidate genes in tumor metastasis.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the jove veterinary review board.
1. Intracardiac injection:
Hemizygous RIP-Tag mice, not homozygous mice, are used in this study. Hemizygous RIP-Tag mice develop pancreatic neuroendocrine tumors around 10~12 weeks of age, while homozygous mice have a shorter tumor latency. Because cell proliferation is required for the incorporation of the viral cDNA into the host genome, 7-week-old RIP-Tag; RIP-tva mice with hyperplasic pancreatic β cells are used. Please note that most normal β cells are not proliferating in adult mice.
Figure 1: Placement of intracardiac injection. Anatomical landmarks are shown with horizontal dashed lines on the sternum (outlined in white). On the mouse's skin, the sternal notch and xiphoid process serve as landmarks, and the needle is inserted 1 mm away from the mid-sternum and slightly left (anatomical) of the sternum. Please click here to view a larger version of this figure.
The authors have nothing to disclose.
Insulin Syringes | BD | 329461 | |
RCASBP-Y DV plasmid | Addgene | 11478 | |
RCAS-RNAi plasmid | Addgene | 15182 | |
DMEM | Corning | 10-013-CV | |
Penicillin-Streptomycin solution, 100x | Corning | 30-002-CI | |
Superfect | Qiagen | 301305 | |
0.45 mm Nalgene Syringe Filters with PES Membrane | Thermo Scientific | 194-2545 | |
Synaptophysin | Vector Laboratories | VP-S284 | |
VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) | Vector Laboratories | PK-6101 | |
AmpliTaq DNA Polymerase with Buffer II | Life Technologies | N8080153 | |
PBS-/-, 1x | Corning | 21-040-CV | |
Superfect | Qiagen | 301305 | |
Synaptophysin | Vector Laboratories | VP-S284 | |
L-glutamine, 100x | Corning | 25-005-CI | |
Fetal bovine serum | Atlanta Biologicals | 25-005-CI |