Here’s an insider’s view on the important gene gun technology we’re using in our science advancement project on an RNAi-based biopesticide. [See previous post.]
The details come from Jeffrey Staub, Ph.D., a TechAccel science advancement program manager, with deep expertise with biolistic transformation via the gene gun. Jeff is also the founder, president and chief science officer of a startup biotech company, Plastomics Inc., which is using gene gun technology to improve crops.
Prepared tobacco leaves waiting to be “shot.”
Gene guns have been in use in plant transformation since the mid-1980s, according to Jeff and Wikipedia. The technology works a lot like a very small and controlled shotgun.
The door is shut; we’re waiting for the chamber to reach the required vacuum level to allow the shot to go off. Note our researcher has his finger on the “trigger.”
To start, you’ll coat micro-sized pellets of gold with the DNA that you want to inject into the target plant. They are on a microcarrier disc, at the top of the chamber.
The plant – we’re using tobacco leaves – is set up as the target at the bottom of the chamber.
First, a vacuum is pulled in the chamber to clear the way for high-pressure helium. Then, when the trigger is pulled, the pressure shoots the DNA-coated gold particles into a stopping screen that lets the particles – but not the microcarrier disc – go through.
Some of those particles enter the chloroplasts of the plant leaf. That’s where the biolistic transformation will occur. (And yes, “biolistic” is a kind of play on words, derived from the traditional “ballistics” or science of projectiles.)
The gene gun is a forceful way of inserting the genetic material into the target. To add even more fun, the team is using the gene gun to put the gene for the RNAi into the tobacco’s chloroplasts, rather than the nucleus. This is a new technique with great promise.
After the bio-material has been blasted into the target plant, the leaves are chopped up and placed into a growing medium. In our project, the transforming DNA has an antibiotic-resistance gene included along with the trait gene. The plant bits are put into a growing medium with an antibiotic that selects for transgenic plants. Only the candidate transgenic chloroplasts will grow in this culture.
After the leaves are “shot” with transforming DNA, they are chopped and placed into a medium with antibiotic that selects for transgenic plants. The transforming DNA has an antibiotic-resistant gene included along with the trait of genetic interest.
So now we wait. It will take several weeks to see the results of our bio-blast.
Stay tuned. The next installment will show what happened.
