Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs
| Autor: | Shih Heng Chen, Thomas M. Porter, Amanda R. Mathew, Erica Scappini, Eugenia H. Goulding, Page Myers, Mitzie Walker, Negin P. Martin, Charles Romeo, Lucas Van Gorder, Artiom Gruzdev |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Genetically modified mouse Zygote Transgene General Chemical Engineering Embryonic Development Mice Transgenic Gene delivery Biology General Biochemistry Genetics and Molecular Biology Mice 03 medical and health sciences Transduction (genetics) Host chromosome medicine Animals Blastocyst Zona pellucida General Immunology and Microbiology Lasers General Neuroscience Lentivirus Gene Transfer Techniques Embryonic stem cell Cell biology 030104 developmental biology medicine.anatomical_structure embryonic structures Female |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/58327-v |
| Popis: | Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs. |
| Databáze: | OpenAIRE |
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