Laser-Assisted Lentiviral Gene Delivery: A Technique to Permeabilize Mouse Fertilized Eggs to Facilitate Lentiviral Gene Delivery

Published: April 30, 2023

Abstract

Source: Martin, N. P., et al. Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs. J. Vis. Exp. (2018).

In this video, we demonstrate the laser-assisted permeation of the protective layer of zona pellucida in mouse fertilized eggs for facilitating lentiviral gene delivery. Lentivirus enables the generation of transgenic animals with a gene of interest stably integrated into their genome.

Protocol

1. Perforation of Mouse Fertilized Eggs with XYClone Laser Setup and calibrate XYClone laser according to the manufacturer's recommendation. Other lasers, typically used for in vitro fertilization, can be substituted for XYClone laser to perforate fertilized eggs. Attach the laser controller box wire to the laser apparatus on the microscope. Attach the laser controller box to the computer running the laser software via a USB port. 3.1.3. Plug in the laser controll…

Representative Results

Figure 1: Laser-Treatment of Mouse Fertilized Eggs. Examples of perforating the zona to produce a hole vs thinning of the zona.

開示

The authors have nothing to disclose.

Materials

CD510B-1 plasmid System Biosciences CD510B-1 used to package the lentivirus expressing EF1a-copGFP
Dimethylpolysiloxane Sigma DMPS5X culturing embryos
XYClone Laser Hamilton Thorne Biosciences perforating mouse fertilized eggs
Non-Surgical Embryo Transfer (NSET) Device ParaTechs 60010 NSET of embryos
KSOM medium Millipore MR-020P-5F culturing embryos
Composition of KSOM: mg/100mL
NaCl 555
KCl 18.5
KH2PO4 4.75
MgSO4 7H2O 4.95
CaCl2 2H2O 25
NaHCO3 210
Glucose 3.6
Na-Pyruvate 2.2
DL-Lactic Acid, sodium salt 0.174mL
10 mM EDTA 100µL
Streptomycin 5
Penicillin 6.3
0.5% phenol red 0.1mL
L-Glutamine 14.6
MEM Essential Amino Acids 1mL
MEM Non-essential AA 0.5mL
BSA 100

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記事を引用
Laser-Assisted Lentiviral Gene Delivery: A Technique to Permeabilize Mouse Fertilized Eggs to Facilitate Lentiviral Gene Delivery. J. Vis. Exp. (Pending Publication), e20995, doi: (2023).

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