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

Samenvatting

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

1. 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.
   1. Attach the laser controller box wire to the laser apparatus on the microscope.
   2. Attach the laser controller box to the computer running the laser software via a USB port. 3.1.3. Plug in the laser controller and switch it on.
   3. Looking through the eyepiece, perforate a test sample (e.g. dry-erase markings on a glass slide).
   4. Use a small screw drive....

Resultaten

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

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Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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
<strong>KSOM medium</strong>	Millipore	MR-020P-5F	culturing embryos
<strong>Composition of KSOM:</strong>			mg/100mL
NaCl			555
KCl			18.5
KH2PO4			4.75
MgSO4&nbsp;7H2O			4.95
CaCl2&nbsp;2H2O			25
NaHCO3			210
Glucose			3.6
Na-Pyruvate			2.2
DL-Lactic Acid, sodium salt			0.174mL
10 mM EDTA			100&micro;L
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