Generation of a Murine Model with a Central Nervous System Infection

프로토콜

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Infection Procedure

1. Prepare nonadapted DENV2 (strain PL046) stocks9 (originally obtained from the Centers for Disease Control in Taiwan, ranging from 2.5 x 10⁷ to 1 x 10⁹ PFU/mL).
2. Dilute the virus stock to 1 x 10⁶ PFU with Roswell Park Memorial Institute (RPMI) 1640 medium to a total volume of 40 μL.
3. Fill one 0.3 mL syringe fitted with a 30 G needle with 10 μL (2.5 x 10⁵ PFU) of diluted virus and fill another 0.3 mL syringe fitted with a 30 G needle with 30 μL (7.5 x 10⁵ PFU) of diluted virus.
4. Hold the 7-day-old ICR (Institute of Cancer Research) suckling mouse and conduct the following steps.
   1. For the intracerebral injection, hold the mouse in a prone position by pressing the auricle between the index finger and thumb, and intracerebrally inject 10 μL of diluted virus into the lambda area, the point at the intersection of the sagittal and lambdoid suture.
   2. After the intracerebral injection, hold the mouse in a supine position using the index finger and thumb, and gently inject 30 μL of diluted virus intraperitoneally into the murine abdomen.
      NOTE: To avoid cannibalism, 75% alcohol is used to create a temporary loss of the mother's olfactory sensation. Additionally, covering the litter with their mother's stool and urine is suggested.
5. Lay the suckling mice back in their cages and wait for 5 min to check their safety poststimulation with the avidity of mice, including walking and mother milk sucking.
   NOTE: Most often, mice demonstrate normal activity poststimulation, and there do not seem to be any harmful effects following the technical challenge.
6. Evaluate the daily progress of the mice in terms of body weight (by using a microgram balance), acute viral encephalitis-like disease (by disease scoring as described in section 2), and survival rate.

자료

Name	Company	Catalog Number	Comments
Roswell Park Memorial Institute 1640 medium (RPMI)	Gibco	11875-085	Diluting virus
0.3-mL Insulin Syringe	BD Ultra-Fine­II	328838	Intracerebral and intraperitoneal injection
Microbalance	METTLER TOLEDO's LabX	AL104	Weight mouse
Non-adapted DENV2 (strain PL046)	The Centers for Disease Control of Taiwan	-	Infect mouse
Institute of Cancer Research (ICR) suckling mouse	BioLASCO Taiwan Co., Ltd	-	Our murine model