The strategy for cloning insulin was conceptually straightforward, although not technically easy. First, one had to isolate the messenger RNA from human insulin-producing cells. Second, this messenger RNA was reverse-transcribed back into DNA, usually called complementary DNA. This piece of complimentary DNA would only contain information on insulin, unlike DNA isolated from a cell, which would contain information on all the genes in an organism. Finally, this new piece of complimentary DNA was inserted into a new cell. For the new cell, Ulrich used a bacterial cell called E. Coli. If everything worked properly, the E Coli would then start producing human insulin.
Every step had significant roadblocks.
Isolating the messenger RNA from human insulin producing cells was hindered in two ways. The challenges here reflected those that the Toronto team faced 50 years before, when they first tried to isolate insulin from the pancreas. First, the insulin-producing cells make up only a tiny fraction of all cells inside the pancreas, less than 1%. Second, the pancreas produced an enzyme, called RNAse, which rapidly degraded any RNA present.
The first step was to develop an inhibitor to the RNAse. William Rutter’s laboratory first developed a method of inhibiting RNAse.
Ullrich started to tackle the problem of cloning human insulin by first working on rat insulin. In order to get enough starting material, he needed to use the pancreas from 200 different rats. Once he isolated the rat insulin messenger, he converted it into complementary DNA then inserted it into a new vector (a piece of DNA that makes control easier) inside of E. Coli. A vector is a kind of standardized wrapper for a cloned gene. By using a vector, one could treat genes as truly interchangeable parts inside of E Coli. Cloning the rat insulin gene was a tremendous accomplishment, but there was a big bump in the road.
Recombinant DNA, moving genes from one organism to another, was a revolutionary technology. Some feared that this technology could be dangerous. What if someone cloned human insulin gene into E. Coli? E Coli was a bacteria native to human’s intestinal tract. It has probably been in every human since the origin of the species. Insulin is a protein that only occurs in higher life forms, it had never been found in a bacteria. If an E Coli strain were artificially produced that made insulin, what would happen if that strain escaped from the laboratory and populated itself in the intestinal tract of a human. Would the human suddenly die of hypoglycemia? Would we be creating a super bug, capable of killing all humans on the planet? No one knew for sure, but there were knowledgeable people on both sides of these debates.