Generation of transgenic mice

We propose you four differents technique about the transgenic mice:

1. transgenic mice by classical DNA injection
2. transgenic mice by BAC injection
3. transgenic mice by zing finger nucleases injection
4. transgenic mice by TALEN injection

For those techniques, it includes in the service:

preparation of the construct for the microinjection, isolation of fertilized oocytes, preparation of donor and recipient mice, microinjection of DNA / RNA into the pronucleus, transfer of injected oocytes into the oviduct of pseudopregnant recipient mice, keeping offspring up to weaning.

Delivery of a minimum of 2 transmitting founders (expression of the transgene not guaranteed)

Potential drawbacks

• the generation of founders indicates that the microinjection was successful, but does not prove that the transgene is correctly expressed
• lack of expression of the transgene might indicate problems in the DNA construct
• lack of founders, in the presence of a positive control injection experiment, might indicate that the transgene interferes with mouse development

Procedure and request form

Generation of transgenic mice by classical technique and BAC injection

Microinjection of DNA / BAC into the pronucleus
• Preparation of the DNA construct (provided by the investigator) for injection
• Recovery of the fertilized oocytes from mice
• Production of donor and recipient mice
• Microinjection of the DNA construct into the male pronucleus of fertilized oocytes
• Transfer of the manipulated embryos into the oviducts of pseudopregnant recipients
• Generation of transgenic lines under controlled conditions
• Delivery of the founders to the investigator
 

Generation of transgenic mice by ZFN or TALEN

Zinc Finger Nucleases (ZFN) are type II enzymes in which the DNA recognition site is separated from the nuclease site. The Zinc finger domain is made of three modules of zinc fingers. The nuclease domain is a recombinant FokI nuclease that functions as a dimer. Therefore, a full ZFN site is composed of a pair of Zinc Finger Nucleases binding to anti-parallel DNA strains and are close enough to be activated. Using this technique we can rapidly generates Knock-outs and Knock-ins

Microinjection of mRNA into the pronucleus and cytplasm.
• Preparation of the mRNA construct (provided by the investigator) for injection
• Production of donor and recipient mice
• Recovery of the fertilized oocytes from donor mice (C57Bl/6N)
• Microinjection of the mRNA construct into the male pronucleus and cytoplasme of fertilized oocytes
• transfer of the manupulated embryos into the oviducts of pseudopregnant recipients
• Generation of transgenic lines under controlled conditions
• Delivery of the founders to the investigator