Transfecting Insect Cells, Pt. 1

Howdy everyone! Guest writing for the TransMission is one of our field application specialists, Dr. Bees… which is a nom-de-plume, of course! I’ll be doing a series of insect-themed posts, starting the month of February for you love bugs and valentines, that includes transfection solutions that Mirus has developed for insect cells.

In Part 1, we begin by introducing the cast of characters and the critical cell biology of insects. In Parts 2 and 3, I will cover baculovirus and baculovirus expression vector systems (BEVS). And, in the final installment, we will bug out on how BEVS can be used to produce adeno-associated viruses (AAV).

Without further ado, let’s meet our first insect friend…

Drosophila melanogaster

Click to hear pronunciation.

This common fruit fly is a model organism familiar to many geneticists. Much of the Drosophila research is done in vivo, however, cell lines including the S2 line, are used for protein production.¹

Trichoplusia ni

Click to hear pronunciation.

The cabbage looper, a moth of the Noctuidae family, gains its name from being a pest to many vegetables, particularly cruciferous vegetables like cabbage. The High Five™ cell line is derived from an ovarian cell of a cabbage looper moth. It is commonly used for viral or recombinant protein expression.^2,3

Spodoptera frugiperda

Click to hear pronunciation.

This is another Noctuidae moth that is a commercial crop eating caterpillar. The Sf cell lines, namely Sf9 and Sf21, were derived from the ovary of an adult moth. These lines are often used for recombinant protein and virus research.⁴ Of note, the Sf9 line is a clone of Sf21 isolated at Texas A&M University, the alma mater of yours truly.

How Insect Cell Lines Are Different from Mammalian Cell Lines

While insects are animals, they are quite different from mammals where most of our cell lines come from: CHO, HEK, BHK, MDBK, Vero etc. Insects are exothermic, so their cells tend to thrive and grow at lower temperatures, 25°C vs. the mammalian temperature of 37°C. Insects have chitinous exoskeletons with more open circulatory systems containing hemolymph, which is analogous but not the same as blood, so their growth media needs differ as well. Most insect cell lines can grow as adherent monolayers or in suspension with or without serum, respectively. Insect cells are not vulnerable to viruses that plague vertebrates (such as adenovirus or lentivirus), having their own set of viruses that we will cover later. With these factors in mind, Mirus has developed a transfection reagent specific to the unique biology of insects, TransIT®-Insect.

In the examples below, TransIT®-Insect was used to effectively transfect a luciferase-encoding plasmid in the cell lines we talked about here: S2, High Five™ and Sf9. Of note, luciferase is an enzyme found in fireflies, a.k.a. lightning bugs (which, by the way, are beetles and not bugs).

TransIT®-Insect Outperforms Competitor Transfection Reagents. Insect cell lines (A) Sf9, (B) High Five™, and (C) Drosophila S2 cells were transfected in 96-well plates with 0.1 µg of a luciferase expression plasmid driven by an hr5 enhancer/IE1 promoter using the designated reagent at the indicated reagent-to-DNA ratios (µl: µg). Luciferase expression was measured at 48 hours post-transfection. Sf9 and High Five™ cells were cultured and transfected in serum-free media formulations; S2 cells were in serum containing medium.  Error bars represent the standard error of the mean for triplicate wells.

 

TransIT®-Insect Yields Increased Protein Expression Over Time. Insect cell lines (A) Sf9, (B) High Five™, and (C) Drosophila S2 were transfected in a 96-well plate with 0.1 ug of a luciferase expression plasmid driven by an hr5 enhancer/IE1 promoter using the TransIT®-Insect Transfection Reagent at a reagent-to-DNA ratio of 2:1 (µl: µg). Luciferase expression was measured at three time points, 24, 48 and 72 hours post-transfection.  Sf9 and High Five™ cells were cultured and transfected in serum-free media formulations; S2 cells were in serum containing medium. Error bars represent the standard error of the mean for triplicate wells.

So, if you’re venturing into the six-legged world and/or transfection is bugging you, please contact Mirus Bio and learn how TransIT®-Insect can metamorphose your research from caterpillar to butterfly!