Traditional transgenes are defined here as transgenes cloned into standard bacterial vectors. These transgene constructions, not including the vector, are usually less than 10 kb. These constructions are usually coding regions fused to promoter elements. If you are attempting to express cDNA sequences, it would be helpful to include an intron and a good polyadenylation signal. The design of these transgenic constructs should include a means to isolate the transgene from the plasmid backbone. The purity of the transgene fragment to be injected is critical for efficient generation of transgenic mice. In order to ensure that the transgene fragment is "Microinjection Quality", the GERM Core will isolate the transgene unless the investigator has a specific reason to isolate the fragment themselves.
What Will Happen
- Investigator will submit a request form through iLabs. An account and IACUC protocol number must be provided at this time.
- The investigator will generate their targeting construct. The GERM Core does not provide construct generation as a service. However, we will review the overall design if requested. The investigator is asked to follow the protocol below for sample submission:
- Digest 100 ug of plasmid with the appropriate restriction enzyme(s) in a 300 ul reaction volume.
- Run an aliquot of the digest on an agarose gel and photograph the results.
- Indicate on the photograph the band to be isolated along with the size of the fragment.
- Attach photograph and Eppendorf tube containing the rest of the digest to a printed out request form and deliver to the GERM Core.
- Note: The transgene must clearly separate from the plasmid backbone during electrophoresis. For example: If the backbone is 2.9 kb and the transgene is 3.0 kb, they will not separate on a normal gel. Additional enzymes would need to be used to allow at least a 1 kb difference between the fragments. If you can't digest the backbone free from the transgene, you may have to redesign.
- The GERM core will use agarose gel electrophoresis to isolate the desired DNA fragment.
- The Core will perform DNA purification, quantification, QC, and preparation of high-quality DNA in microinjection buffer.
- The core will prepare and collect 1-cell embryos from C57BL/6J, C57BL/6N, or FVB/NJ after superovulation and perform microinjections of 150-200 embryos. The surviving embryos will be transferred to pseudo pregnant females.
- Live-born founder animals will be held by the GERM Core until 10-14 days of age and subsequently transferred to the Investigator.
- Investigator will genotype and identify the successfully targeted founder mice and subsequently mate them to identify germline transmission.
What is Expected
- The number of live-born pups can significantly vary depending on the transgenic construct with very few to 50+ pups born.
- A minimum of 5 live-born offspring. If 5 live-born offspring are not produced, the microinjection will be repeated.
- Due to random integration, the expression pattern and levels of a transgene can significantly very from founder to founder. Additionally, random integration can alter the expression of an endogenous gene, causing phenotypes not directly attributable to the transgene itself. Therefore, independent lines should be derived from several founder animals and screened for expected transgene expression levels and patterns and expected phenotypes. Generally, a phenotype associated with the transgene, and not a disrupted endogenous gene, should be observable in multiple founder lines.
- Founder animals can be mosaic and may carry several integration sites that can be independently transmitted to the next generation. Variability in phenotype and transgene expression level may be due to various sites segregating within a transgenic line.