If large fragments of genomic DNA averaging 100-300 kb need to be microinjected, then Bacterial Artificial Chromosome (BAC) clones harboringlarge genomic DNA fragments (100-300 Kb) can allow faithful tissue specific expression inatransgenic setting.However, due to their size, the amount of DNA that can be microinjected into an embryo is significantly less than a standard transgene and the constructs can easily shear, reducing theefficiency of generating transgenic mice. Integrated copy number is also typically less than a standard transgenic construct. Successful generation of BAC transgenics is highly dependent upon the designing and preparation of the DNA.
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 is responsible for preparing BAC DNA (linearized or circular) for microinjection. The GERM Core does not provide preparation services; however recommended protocols and kit suggestions will be provided. It is critical that a kit designed for large constructs is used to minimize shearing.
- The investigator is also responsible for quantifying the BAC DNA preparation. DNA must meet the following parameters:
- 260/280 of 1.70 to 2.0
- Digestion of an aliquot of BAC DNA with a frequent cutter, e.g. BamHI, and run on a 0.8% agarose gel. A distinct, predicted banding pattern based on the BAC sequence (a BAC fingerprint) should be visible.
- If linearized, run DNA on a pulse field gel. You should see a single, sharp band of the expected size.
- Do not freeze BAC DNA as it will shear after thawing.
- Investigator will deliver the BAC DNA prepared to the GERM Core with a copy of the iLab request form attached 1-7 days prior to the scheduled microinjection. BAC DNA will degrade/shear more rapidly than a standard plasmid.
- The GERM core will store the resuspended DNA at 4ºC until the microinjection day.
- On the morning of microinjection, the GERM core will repeat the 260/280 measurements and dilute it to 1ng/ul into microinjection buffer.
- The core will prepare and collect 1-cell embryos from C57BL/6J, C57BL/6N, or FVB/NJ after superovulation and perform microinjections to 150-200 embryos. The survival 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 then transferred to the User Investigator.
- Investigator will genotype and identify the successfully targeted founder mice and subsequently mate the founders 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 is guaranteed. 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.
- BAC DNA can easily shear and only fragments of the BAC DNA may integrate into the genome. If a BAC harboring genomic DNA from one inbred strain (e.g. 129) is microinjected into another strain (e.g. FVB) PCR-based screening of microsatellite markers or SNP variant screening can be used to identify regions of BAC DNA integrated. If a BAC harboring genomic DNA from human is used, PCR-based screening for human sequences, Sanger sequencing for human sequences, or Southern blotting for human repeat sequences (e.g. Alu elements) can be used to identify regions of BAC DNA integrated.