Designing projects
If desired, the TMF is here to help you design your transgenesis project, helping in the molecular cloning of the vectors (strategies design, training of staff and students). We can also help you with writing grants for the project.
Pronuclear injection for classical transgenesis (gain of function)
In classical transgenesis, a transgene is injected directly into a fertilized oocyte. The transgene can be randomly inserted into the genome (usually in multicopy). The transgenic animal will gain a function by overexpressing the transgene.
Examples of application:
Expressing a reporter gene to study a pattern of expression of a promoter,
Expressing a protein of interest according to a specific and controlled time/space pattern,
Expressing a dominant negative (an altered version of a gene which is going to interfere with the native protein)
Homologous recombination (loss of function)
A vector for homologous recombination is electroporated into Embryonic Stem (ES) cells. A rare event of homologous recombination can be selected. The ES cells are then injected into blastocystes of mice to give birth to chimeras (containing cells derived from the original blastocyste and cells derived from the recombinant ES cells). These chimeras can be bred and give birth to recombinant animals.
Examples of application:
Interrupting a gene of interest (Knock-Out or KO) and study the effect of the absence of the protein,
Replacing a sequence of interest by a slightly modified sequence (punctual mutation for instance) or
Inserting an exogenous sequence into a controlled region of the genome (ROSA locus).
These two last examples lead to a Knock-In, which can lead to a gain of function without the possible non-specific effects dependant from the random insertion of the classical transgenesis.
Rederivation
Lines obtained from an outside facility need to have a health status matching the SPF status of the facility. If not, the lines will be quarantined and rederived. Embryos are collected and re-implanted into foster mothers at the TMF. As pathogenes are not transmitted to the eggs, the newborns are clean.
Genotyping
Genotyping of the transgenic lines is a service that can be provided by the TMF. The standard procedures to genotype mice are Dot blots and PCR. Other techniques can be performed as well. (Southern blots)
Colony management
The first two generations of a new line are systematically bred within the TMF, by the TMF personnel. The further breeding can also be taken care of by the TMF if necessary.
In vitro fertilization
Sperm and eggs can be extracted and fertilized in vitro, then re-implanted in a foster mother. IVF can be used when mice do not reproduce well naturally, or to enhance a production very fast for a specific purpose.
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