Pronuclear injection of fertilized eggs is the most common and convenient way to make transgenic mice. This method typically results in multiple transgene copies inserted at a single locus. However, it is important to be aware of some of the shortcomings of the technique. For example, late integration of the transgene can results in mosaic founders which can, in some cases, negate germ line transmission of the transgene. About 1 in 10 insertions result in a phenotype due to disruption of the host genome. Expression levels do not correlate well with copy number, and not all integrated transgenes are expressed, or expressed in a consistent manner between lines. In addition, expression may be progressively extinguished due to methylation of the transgenic locus during breeding of a given line.
The typical transgenic construct includes an enhancer/promoter to direct expression in the tissue of interest, the coding sequence of the gene of interest, and a poly-adenylation signal sequence. A splice donor, intron and splice acceptor sequences between the coding sequence and the poly-adenylation signals can increase expression. It is important that the construct be designed such that the transgene insert can be purified away from the plasmid backbone by gel purification.
You will need a strategy to detect the transgene in mice, in order to identify founder mice and the transmission of the transgene. While PCR-based approaches are a very sensitive means to this end, genomic-southern blot also suffices to detect single copy transgenes, and is a useful means to assay for gross rearrangements of integrated transgenes.
Which strains of mice?
At present, the facility is restricted to FVB/N, a robust strain with high fecundity and zygotes that are easy to inject and C57BL/6J upon request. Generation of C57BL/6J transgenics necessitate a concomitant increase in pricing due to reduced yields of embryos for microinjection.
Bacterial Artificial Chromosome (BAC) Transgenes
BAC transgenes have numerous advantages over plasmids in that they frequently yield reproducible levels of expression, are much less susceptible to epigenetic inactivation, and give expression proportionate to copy number. BAC clones often recapitulate normal spatial and temporal expression of the gene of interest. BAC clones can now be manipulated by "recombineering" approaches to insert or delete specific sequences.
A number of mouse and human genomic BAC libraries have been end-sequenced and tiled on the genome (see, or e.g. GENOME UCSC) and BACs of interest can be readily obtained from such sources (e.g., BACPAC CHORI). With rare exceptions, the size of a BAC clone is typically sufficient to include the entire gene and relevant regulatory sequences. BACs are relatively stable in E. coli and easy to purify. While BAC-based vectors can be used for transgenesis, they are more difficult to inject and typically yield a lower percentage of transgenic offspring.
Investigators need approval from their institute for the establishment of any mouse model to be generated by the Facility. Work will not proceed until a relevant, active Protocol number has been received.
The facility operates on a first-come first-served basis. For transgenics, a minimum of 7 weeks is required from commencement of injection before delivery of ear lobe or tail biopsies for analysis. This does not include wait times related to service demands.
What the user will provide
We strongly encourage users to discuss the particulars of any given transgenic construct prior to injection (or prior to initiation of construction for those less familiar with the technique). This is necessary in order to anticipate potential problems, such as embryonic or postpartum lethality which can be elicited by some transgene products.
A cost center or PO number provided to the Core Facilities manager.
A minimum of 50 micrograms of plasmid DNA prepared to high quality by Qiagen maxi-prep and restricted to permit isolation of the insert from the plasmid backbone.
A photograph of an aliquot of this digest that has been run on a gel with appropriate molecular weight markers and an indication of the bands corresponding to the plasmid backbone and to the transgene to be injected.
A demonstration that 1 copy of the transgene can be detected by Genomic Southern blot or by PCR approaches in a background of mouse genomic DNA.
The user will isolate DNA from ear lobe or tail tip biopsies, use this to identify transgenic pups, and relay this information to the Core. The facility will house all pups for a maximum of 10 days post weaning date to permit this analysis, after which time per diem charges will be levied to the user.
What the transgenic core facility will do
We will gel-purify the plasmid insert, inject the insert into a minimum of 100 fertilized FVB embryos with the goal of delivering at least 3 transgenic offspring per injection.
We will house the offspring from the injections until approximately 4 weeks after birth, and provide you with ear-lobe or tail-tip biopsies for identification of transgenic offspring.
We ask that you acknowledge contributions of the University of Ottawa, Faculty of Medicine, Transgenic Core Facility in seminars and publications.
Please note: We will endeavor to deliver as many transgenic offspring as possible, however, due to the nature of the process, we can offer no firm guarantees. To this end, consultation with the Core manager and/or director is strongly encouraged prior to any submission for services.
Cryopreservation, Cryorecovery and Rederivation
A major challenge to any animal facility is the judicious management of space. However, after initial experimentation, mouse lines are often kept breeding in perpetuity in case they are needed at some future date. This not only consumes limited housing, but also represents a considerable cost to the investigator. To help alleviate these issues, the facility has initiated a cryopreservation service in order to archive mouse lines.
For embryo cryopreservation, C56BL/6, FVB or CD-1 females will be crossed with males to be provided by the user; additional strains may be considered, with any additional costs to be borne by the investigator. A minimum of 7, and preferably 10, males of proven fertility are necessary. A minimum of 250 viable zygotes will be harvested from these matings, frozen and stored by the core. Note that we cannot offer an absolute guarantee of recovery from these stocks. Users are encouraged to test frozen stocks for particularly critical lines.
The core now also offers sperm cryopreservation. This is a cost-effective and efficient means of archiving genetically modified strains, and has the advantage of requiring only a few mature males.
Recovery services for cryopreserved embryos and sperm and rederivation of contaminated mouse lines are also routinely offered.
In addition, we are often able to revive endangered lines through in vitro fertilization and can produce large cohorts of mice using enhanced superovulation methods.
Embryonic Stem Cell Culture and Production of Germ-Line Chimeric Mice
The core offers recovery services for the derivation of engineered embryonic stem (ES) cells. We provide a ES cell culture “package” consisting of embryonic fibroblasts, ES cell media and germ-line competent ES cells and we host a ES cell suite for the culture of cells and isolation and propagation of clones. We also carry a number of vectors that may be of use for the derivation of targeting constructs, and are available to assist in the design of targeting constructs. Finally, the core provides assistance for the production of KO mice via blastocyst microinjection of mutation-targeted cells.
The core provides services to receive or ship embryos as an efficient means of distributing lines.
The core can produce microinjection pipettes according to desired characteristics for injection of mouse embryos, Hela cells, Drosophila, etc.