Transgenic Mouse Production
The transgenic mouse has assumed increasing importance as an experimental system in recent years. The uses of this technology are numerous, but they include studies of normal and altered gene function, analysis of elements responsible for developmental and tissue-specific gene expression, and the production of animal disease models. Transgenic mice are generated by injection of a recombinant DNA construct directly into the male pronucleus of a fertilized egg. The injected eggs are transferred to pseudopregnant females and the resultant pups are tested for the presence of the transgene by Southern blotting of DNA from tail biopsies. Multiple copies of the transgene integrate into a single, unique site usually in a head to tail array. Therefore, each transgene positive founder will represent an unique integration site which may affect transgene expression or regulation. The Core will inject an individual transgene construct up to three times with the goal of producing up to 15 potentially transgenic pups from each injection session. Tail DNA will then be provided to the investigators for analysis. Once transgene positive pups are identified, they will be given to the investigator and placed in the mouse facility of their choice.
1) General Considerations
Transgenic mice have been produced using a wide variety of genetic elements including specialized promoters, cDNA constructs and reporter genes. In theory, various genetic elements can be utilized to generate transgenic mice and this section is not designed to debate the pros and cons of any specific system. Instead, we have listed various factors which affect the success of transgenic experiments regardless of the nature of the DNA construct used. If you have questions concerning the use of specific genes or promoter sequences in your experimental design either consult the literature or contact the TMCF office directly.
Effect of Prokaryotic Sequences
Although the presence of prokaryotic sequences do not appear to affect integration frequency, they have been observed to severely inhibit transgene expression in a number of cases. Therefore, it is advisable to remove all vector sequences from the transgene construct prior to injection.
Use of cDNAs
Although it is possible to use cDNA sequences to generate transgenic mice they tend to give much lower levels of expression than when the corresponding genomic sequences are used. The apparent cause of this difference is the presence of intronic sequences in the genomic clones. Therefore, when using cDNA-based constructs it is often advisable to construct a minigene containing one or more introns.
Purity/Concentration of the Injected DNA
The purity and concentration of the injected DNA is one of the most critical factors to the success of transgenic experiments. It must be free of all particulate matter and dissolved in the appropriate buffer at a concentration of 1-5 mg/ml. Due to the critical nature of this step the core will purify all DNA for microinjection. It is the investigator's responsibility to provide high quality DNA which has been fully digested with the appropriate enzymes such that the construct to be injected is free of all vector sequences.
2) Instructions for Use of the Transgenic Service
A) Schedule a preliminary appointment with the TMCF director. At this time we can review your experimental design and we can help you with any problems which you may have.
B) Obtain approval from the animal care committee for your project. This is crucial. You cannot have animals without prior approval. Therefore, the core cannot begin your experiments until this approval is given. The necessary forms can be attained from the Animal Care and Use Committee (phone # 49163). The core has approved protocols on file for generating transgenic mice. You simply need to write a protocol for what you will do with the mice once you receive them
C) Obtain approval from the Institutional Biosafety Committee (IBC) for the use of recombinant DNA in your research. Again, this must be approved before we can begin your experiments. These forms are available from the IBC office (phone # 41063).
D) Turn in a Transgenic Mouse Requisition Form.(FORM A).
E) Provide the core with digested DNA. Include a gel photo indicating which band contains your construct.
F) Provide a scheme for identification of the transgenic pups, including the results of a control experiment. This is for your protection. There is nothing more frustrating than having potentially transgenic pups sitting in the animal facility and not being able to identify which are positive. This is not always a trivial experiment and the conditions for transgene identification should be worked out well in advance of receiving potentially transgenic tail DNA.
G) Complete a billing form (FORM B). We will give your experiment a project code number. However, please give a code name to your experimental construct where we ask for a description of the construct. This will allow you to identify an individual experiment upon receiving your bill. You will be billed for services at the end of the month in which your experiments are initiated, with the actual charge being incurred 2-3 weeks later.
H) The Core will inform you when your experiments are performed and will send you a data sheet when pups are born. After weaning you will receive tail DNA from the potentially transgenic pups and they will be transferred to your animal account number. At that point you have 2 weeks to identify the mice and inform the TMCF which mice are transgene positive. Please complete FORM C at this time and bring this along with a copy of the results to my office. The positive pups will then be transferred to the animal facility of your choice. NOTE: Because of the limited space within the core, all animals must be identified and leave the facility within the 2 week time limit.
3) Experimental Time Line
Listed below is a time line for the experimental phase of making transgenic mice. The time it takes from the planning stage to the initiation of your experiments will depend in large part on how long it takes for you to obtain all of the required approval forms. Therefore, it is never too early to start this process. The animal approval process often takes 2-3 months between receiving the forms from animal care to final approval, so do not wait until you have DNA ready for us to inject before you begin this process. Again, we cannot start your experiments until we have these approvals.
4) Forms and Protocols
(Adobe Acrobat PDF Files)
Form A. Transgenic
Mouse Request Form
Form B. Rate Charge
Form
Form C. Transgenic
Mouse Screening Results
Southern Blotting Protocol