Services

The services provided by the Neurotransgenic Laboratory are listed below :

Consultation
The Lead Scientist and if necessary the Director of the Neurotransgenic Laboratory will be available to discuss, plan and outline the
experimental steps that are necessary to express a transgene in a mouse. If requested a scheme can be produced and presented to
the initiating investigator for review and approval before the project is started. A consultation fee of $200 will be assessed.

Target Vector Construction
The necessary experimental procedures will be preformed by the staff of the Neurotransgenic Laboratory. Transgenes that are not readily available to the facility will have to be obtained in conjunction with the initiating investigator. Readily available transgenes are fluorescent
proteins (GFP, mRFP, YFP etc.), recombinases (Cre, CreER) and the mammalianized reverse tet transactivator, mrtTA.

Homologous Recombination into Bacterial Artificial Chromosomes
Once the targeting vector plasmid has been completed, our facility will derive the targeting fragment from this plasmid and gel purify it for homologous recombination. The original BAC, containing the desired driver gene, will be transferred to recombination competent bacteria (DY380, EL250 or EL350). Colonies will be veryfied by PCR and positive clones will be made electrocompetent for transfer of the BAC.
EL250 bacteria contain an arabinose inducible flip-recombinase expression unit, that is used to remove the frt-site flanked neomycin gene
from the recombined BAC. EL350 bacteria contain an arabinose inducible cre-recombinase, that can be used to excise floxed DNA
fragments from the BAC transgene vector.

Preparation of DNA Constructs for Microinjection
Bacteria that harbor the modified BAC transgene will be grown for BAC minipreps and the BAC DNA will be transfered to non-recombinogenic bacteria (DH10b or GeneHogs). Colonies will be verified and positive clones will be grown for large scale BAC DNA preparation. Typically
we will run a CsCl gradient in an Ultracentrifuge to purify the BAC DNA. Before microinjection, the BAC DNA will be dialyzed against microinjection buffer, usually over night and then quantified by spectrophotometry and comparative agarose gel electrophoresis. The concentration of the BAC DNA preparation will then be adjusted to 1ng/microliter with microinjection buffer.

Pronuclear Injection into Oocytes and Implantation
Both, recipient female mice screened for ovulation status and oocyte donor mice treated with hormones for superovulation, will be bred
the day before the microinjection procedure. Recipient females are bred to vasectomized males, donor females are bred to fertile males.
The following day, about 140-180 fertilized eggs are collected from the donors and prepared for microinjection. Successfully injected
eggs are screened for morphological signs of damage, and those embryos that appear viable will be implanted into the infundibulum of anesthesized recipient pseudopregnant females. We typically implant 100-120 embryos into 2-4 pseudopregnant females.

Transfer of embryonic stem cells from recombined clones into blastocysts
Recombined or genetically modified embryonic stem cells will have to be prepared and provided by the investigator. These experiments
have to be planned about two weeks prior to the actual ES-cell transfer into blastocysts. We will prepare the female blastocyst recipients
as well as the blastocyst donors in a similar manner as described for the Pronuclear Injection. Depending on the quality of the embryonic
stem cells, we usually get 2-4 chimeric animals (evaluated by coat colors) that will transmit the altered genetic trait of the ES-cells to their
progeny.

Genetic Screening of Offspring
Three weeks after the pronuclear injection, the recipient females will give birth to pups that can be screened for the presence of the
transgene in their genome. We usually take a very small tail biopsie at P7 to P10, extract DNA from these samples and perform a PCR
reaction with the tail DNA template and transgene specific primers. Samples from the transgene positive pups will be rescreend by an independent lab member for verification. Reconfirmed transgene positive animals will be raised until weaning and then transferred to
the mouse colonies of the individual investigators.