Lentiviral Vector-Based Perivitelline Injection: A Method to Transduce the Gene of Interest Into Fertilized Single-Cell Mouse Oocytes
Published: April 30, 2023
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. 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. Take the prepared glass pipet for collecting eggs and connect it…
Representative 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 overall shape of both pipette types is drawn. The dashed circle highlights the enlarged area of the pipet tip. Pictures of the p…
Divulgazioni
The authors have nothing to disclose.
Materials
| 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 |
Tags
Citazione di questo articolo
Lentiviral Vector-Based Perivitelline Injection: A Method to Transduce the Gene of Interest Into Fertilized Single-Cell Mouse Oocytes. J. Vis. Exp. (Pending Publication), e20994, doi: (2023).