What is Transgenics?
Transgenic technology is a process by which your gene of choice is inserted into the mouse genome by random integration by either pronuclear injection of the DNA into a fertilized mouse oocyte (most common) or alternatively by electroporation into ES cells.
Which strain of mice do we inject?
The Gene Targeting andTransgenic Shared Resource at Roswell Park injects hybrid F2 (C3Hf/HeRos X C57BL/10 Rospd) fertilized oocytes.
What if my laboratory needs our transgene on a different background?
We use the B10XC3H background currently because of the hybrid vigor it conveys. Other strains are known to be unsuitable for transgenics due to a variety of reasons: low oocyte yield, difficult visualization of the pronuclei, easily lysed oocytes upon pronuclear injection, poor implantation rates. Our facility will try to accommodate the investigator’s strain needs. There are multiple possibilities that we can explore:
- You can backcross your mice to the desired strain’s background. Our facility can provide you with consultation on how to do this.
- You can purchase 15 stud males and 15 females/week of the desired strain. Our facility will perform superovulations, timed matings and inject those oocytes. Your laboratory would be responsible for all mouse charges incurred, and at the completion of your project, we would hand all mice over to the investigator’s laboratory.
- It may be possible to purchase ES cells derived from that strain background. If so, your construct would need to be modified to include a selectable marker, and an additional mouse cross would be needed to check for germline transmission of your gene. Please see ES-based transgenics for details.
What are the critical parameters in making a transgenic mouse?
First and foremost is the quality of the DNA injected. We ask the investigator’s lab to follow strict protocols to ensure this. The slightest trace of contaminating agents will be harmful to zygotes, and will cause them to lyse shortly after injection or will prevent them from development at later stages. We ask you to do TWO CsCl gradients as per our protocol. Alternatively, our facility can prepare the DNA for your lab for a fee.
How many injection days does it take to get a founder?
This is variable. We usually suggest a minimum of 2 microinjection days. It may take more or less depending on a number of factors.
What should be of concern when making a transgenic line?
- May result in multiple transgene copies inserted at a single locus.
- Occasionally, late integration results in mosaic founders.
- Multiple independent insertions in a founder occur with low frequency.
- About 1 in 10 insertions result in a phenotype due to disruption of the host genome.
- In addition to insertional mutations, deletions and complex rearrangements of the host genome can occur.
- Expression levels do not correlate well with copy number.
- Not all integrated transgenes are expressed.
- Expression may be lost due to methylation arising during breeding of the line.
- For analysis of transgene function, it is wise to work with three independent transgenic lines because of effects of the host genome on the transgene and of the integration event on the host genome.
- Despite these shortcomings, pronuclear injection is often the quickest way to achieve your goals.