In some ways, transgenes resemble foster kids who are expected to thrive and express themselves after being plucked from one location and deposited in another. They flourish or flounder, depending on the new surroundings. A novel technique now ensures their success in Drosophila, insulating the introduced DNA from the disruptive influences of nearby chromatin.
Working in the lab of Howard Hughes investigator and HMS professor of genetics Norbert Perrimon, postdoctoral researcher Michele Markstein sandwiched transgenes between protective stretches of DNA before inserting them into the germ cells of fruit flies. The resulting animals expressed the transgenes optimally in every tissue tested. The method appears in the April issue of Nature Genetics.
“Classically, transgenes are randomly integrated into the DNA of the host cell, often landing in areas where the surrounding chromatin prevents them from being optimally expressed,” explained Markstein. “We’ve insulated these transgenes, so they’re expressed at high levels, regardless of their integration spot along the chromosome.”
“Using Michele’s method, we can create quality transgenic flies on the first try,” added Perrimon.
Initially, Markstein played caseworker for the transgenes. She searched for a perfect home in the DNA of the host cells, a location where the transgenes would always be nurtured rather than a random residence where they might be silenced.
“Michele tried about 20 integration sites, but none of them proved optimal in every tissue of the fly,” explained Perrimon. If the transgene worked in muscle, for example, it might be silent in the brain.
Markstein decided to take a different approach. Seeking to safeguard the transgenes from trouble in their new homes, she borrowed a trick from the gypsy retrovirus, which infects fruit flies. This pathogen uses insulator sequences to protect its own genetic code from the DNA of the fly. Markstein took these insulator sequences and added them to her transgenes before unleashing them on the germ cells of flies.
“In a sense, the transgene travels with the perfect environment, enabling it to function optimally wherever it lands,” said Markstein.
According to Perrimon, the new method could be applied to other species, though labs must first identify an appropriate insulator sequence, since the gypsy retrovirus is specific to flies. In the short term, the method will advance Perrimon’s goal of creating a massive RNAi library of transgenic fruit flies for use in experiments. Each line of flies will contain a single transgene, coding for a short interfering RNA that disrupts the expression of an endogenous gene.
“The library should be complete in three to four years,” said Perrimon. “These fruit flies will allow us to probe developmental and physiological processes at a systems level in organisms.”
