CFCM generates transgenic mice via lentiviral infections, allowing to produce a large number of transgenic mice that contain a single-copy transgene integrated in actively expressed chromatin regions. It was set up in collaboration to the group of Luigi Naldini of the San Raffaele Telethon Institute for Gene Therapy (SR-Tiget).
The production of transgenic mice via pronuclear injections is relatively inefficient, only 15% of the oocytes injected carry the transgene and half of these express them. The transgene is integrated randomly into the genome in multiple copies (from 1 to 200) implying some disadvantages:
- positional effect (such as disruption of a gene crucial for the life of the mouse or insertion into silent regions of the chromatin);
- dose dependent phenotype due to the number of transgene copies integrated, thereby, the necessity to analyze several founders to confirm the phenotypic effect.
Lentiviral infection bypasses the disadvantages of pronuclear injections:
- The lentiviruses particles infect highly efficiently the oocytes.
70% of the treated oocytes carry the transgene.
- The trangene is stably integrated into the genome in actively expressed genomic regions.
Each founder expresses the transgene.
- The transgene integrates in single copy in different genomic regions.
- Transgene copies segregate generating several murine lines with a single copy of transgene from each founder.
The needle is inserted into the oocyte and DNA is microinjected into the male pronucleus. This technique perturbs drastically the environment of the oocyte.
The viral particles are injected in the subzonal space between the cytoplasmic membrane and the zona pellucida. Subzonal injection does not perturb the environment of the oocyte.
About the service
CFCM will microinject the viral particles into 150 fertilized eggs of FVB or C57BL/6 mice. Embryos will be transferred into foster mothers and pregnancies will be monitored. The investigators will then receive tail biopsies from 14 days old mice for genotyping.
Brown BD, Venneri MA, Zingale A, Sergi Sergi L, Naldini L. Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nat Med. 2006 May;12(5):585-91.
De Palma M, Venneri MA, Galli R, Sergi Sergi L, Politi LS, Sampaolesi M, Naldini L. Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell. 2005 Sep;8(3):211-26.
Amendola M, Venneri MA, Biffi A, Vigna E, Naldini L. Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters. Nat Biotechnol. 2005 Jan;23(1):108-16. Epub 2004 Dec 26.
Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science. 2002 Feb 1;295(5556):868-72. Epub 2002 Jan 10.