Lentiviral Vector-Based Perivitelline Injection: A Method to Transduce the Gene of Interest Into Fertilized Single-Cell Mouse Oocytes

Abstract

Source: Dussaud, S. et al., Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders. J. Vis. Exp. (2018).

This video describes the pronuclear injection of lentiviral vectors into the perivitelline space of a fertilized mouse embryo. The lentivirus contains the transgene, which is incorporated into the genome of the fertilized embryo, eventually giving rise to transgenic mice.

Protocol

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

1. Remove cumulus cells from fertilized eggs.

1. Under a stereomicroscope, use 2 insulin syringes: the first one to hold the oviduct and the second one to tear up the ampulla and disperse fertilized eggs into the hyaluronidase working solution.
2. Take the prepared glass pipet for collecting eggs and connect it to the tubing and a 0.22 μm filter mounted on the mouthpiece to aspirate all the eggs. Collect all eggs and wash them by successive passage into 6 diff....

Results

Figure 1: Preparing the microinjection pipettes and genotyping.
(A) Schematic drawings of microinjection pipettes to highlight main differences between pipets used for DNA or lentiviral vector injections. Left panel: the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Insulin syringe  	VWR	 613-3867	Terumo Myjector
Aspirator tube assemblies for calibrated microcapillary pipettes  	Sigma	A5177-5EA
Hyaluronidase Enzyme 30mg  	Sigma	H4272 	mouse embryo tested
Borosilicate glass capillaries  	Harvard apparatus	GC 100-10
Inverted microscope	 Nikon	  Transferman NK2 5188	Hoffman modulation contrast illumination is required
Micromanipulator 	Eppendorf 	Celltram air