Transgene Injection Information

Transgene Injection Information

 

Transgenic mice are constructed by injecting cloned DNA into fertilized mouse eggs; those eggs that survive are then implanted in foster females to develop to term. The gestation period of the mouse is 19-21 days. Pups are ready for weaning at 3-4 weeks of age, and reach sexual maturity at 6 weeks (females) to 8 weeks (males). The minimum elapsed time between injection of the construct and readiness for breeding of the transgenic founders is 9 to 11 weeks. For most experimental purposes researchers will want to use offspring of a transgenic animal, rather than the founder animal itself.

Several factors can affect the production of transgenic mice. For example, the purity of the DNA construct to be injected is very important to avoid toxic effects to the eggs, and the DNA must be at the proper concentration. The DNA construct must be purified away from vector sequences. Also, some expression constructs (e.g. beta-actin-MyoD) may have activities that are lethal for the transgenic embryos. Another caveat is that if transgene integration occurs after the first round of DNA replication in the single fertilized mouse egg, the mouse will be mosaic for the transgene, i.e. only a subset of cells will carry the transgene, and if the germ cells don't carry the transgene, it will not transmit to the offspring. We will make every reasonable effort to make transgenic mice with your construct, but no facility can guarantee success with every construct.

The GTTF will isolate the transgene DNA from the DNA constructs provided by the investigator, then microinject this into approximately 300 fertilized eggs, transfer the embryos into foster females and monitor the pregnancies. The GTTF will perform the tail biopsies and tag the pups, then transfer the pups and biopsies to the investigator. The investigator will perform DNA isolation from the tail clips, and analyze the DNA by PCR and/or Southern blotting in a timely manner. Pups, including potential transgenic founder animals, will be transferred to the care of the investigator upon weaning (3 weeks of age), who will arrange appropriate Vivarium housing and breeding for them outside of our facility.

If the studies on tail biopsy DNA (with adequate controls) fail to demonstrate the presence of at least three transgenic animals, we will reinject the construct at no additional cost to the investigator. If no transgenic animal is detected after the second set of injections, we will hold a meeting to discuss further plans, such as re-purification of the DNA construct, or repeat injections and analysis of potential transgenic embryos if the DNA construct may result in embryonic lethality.

The investigator should provide a high purity DNA construct, the GTTF will further purify the transgene away from the vector. The investigator must purify DNA from the tail biopsies and analyze the samples to determine which mice carry the transgene, in a timely manner. The pups, including the transgenic founder mice, will be transferred out of the facility to the care of the investigator upon weaning.

Transgenes should include promoter, enhancer, gene to be expressed, splice donor and acceptor and intron sequences, and termination/polyadenylation sequences. Transgenes must be excised from the bacterial plasmid sequences in order to be expressed in mice.