Amaxa, a German transfection technologies shop, said last week that the HudsonAlpha Institute for Biotechnology in Huntsville, Ala., will use its Nucleofector technology as a “core technology” in certain research projects.
The deal also calls for the institute to receive Nucleofector kits at a discounted price in return for an exchange of information with Amaxa.
HAIB researchers will use the technology to develop reproducible, non-viral based, transfection protocols for the National Cancer Institute’s NCI-60 cell lines.
Anita Bansal, a doctoral candidate at HAIB who is spearheading the project, told CBA News that the institute is using the Amaxa technology to optimize transient transfection conditions for the NCI-60 cell lines by determining which of the 93 programs pre-set on the machine will give her team maximum transfection efficiency.
Nucelofector’s 96-well format offers the researchers a high-throughput technology, said Bansal, so that many experiments can be done in parallel.
In addition, Bansal noted that the system also offers a much lower rate of cytotoxicity than chemical transfection — around 10 percent to 20 percent with nucleofection as opposed to 50 percent to 60 percent with chemical transfection.
The HAIB researchers are also doing experiments to take advantage of [an undisclosed vendor’s] two-color technology, said Doug Ross, CSO of Applied Genomics and Bansal’s thesis advisor. He explained that the ability to run 96 samples in parallel using a two-color fluorescent probe readout allows the investigators to get more precise quantitation of about 100 promoters very rapidly in a single cell line.
Amaxa’s Nucleofector technology is used to transfect DNA, siRNA, or small molecules into either the cytoplasm or the nucleus of target cells.
Cells of interest are harvested and combined with a mixture of cell type-specific Nucleofector solution and supplement. The DNA or siRNA are added and the mixture is transferred to a cuvette, which is placed in a Nucleofector device where electric parameters and the cell type-specific solution are combined.
When the cuvette is removed, it is rinsed with 500 µL of culture medium, and the nucleofected cells are transferred to a culture dish. Depending on cell type, expression is detectable within three to eight hours, and analysis can be done the same day.
Focus on Promoters
According to Ross, the Amaxa technology was able to isolate around 200 promoters that were of interest because of prior research that he did in cancer-derived cell lines.
He added that transfection conditions for the use of Nucleofector technology have been established for about 25 to 30 different cell lines, and that HAIB researchers have done experiments to optimize protocols that use Nucelofector technology and are now generating data that look at about 200 different promoters across these 25 to 30 cell lines.
In the future, Bansal said, she and her colleagues are planning to use the technology to create stable cell lines for knockdown purposes using siRNA. “We want to see how much siRNA is needed to knock down the genes and we are trying to do multiple knockdowns in a single cell line,” she said.
Ross mentioned that the investigators are exploring two pathways: an interferon response pathway and an oxidative stress pathway. “The question we are asking is, ‘How much of the perturbation of those pathways is reflected in promoter activity of those genes?’ ”
He added that the researchers are interested in whether cancer induces constitutive activity of these pathways that is reflected in the controlled dysregulation of genes.
“It still remains a question as to whether … we have established dysregulation of these pathways at the promoter level,” said Ross. “But in principle, if that turns out to be true, then Amaxa’s technology could be combined with promoter technology as a screen for compounds that perturb these pathways.”
“Amaxa’s technology could be combined with promoter technology as a screen for compounds that perturb these pathways.”
Targeting Primary Cells
Ultimately, those who will benefit the most from this agreement are investigators working with any of the NCI-60 cell lines, said Ted Maker, US vice president of marketing for Amaxa. Once HAIB completes this initial project, the institute would like to further develop these protocols, because it is doing the basic work, Maker said.
Once the protocols are made available, Amaxa can share that information with anyone doing any kind of cancer research, said Maker. He added that anyone who wants to use these cancer cell lines will now have an easy way to work with them.
“We are interested in enabling researchers to start using different cell types that are relevant to disease,” Maker said. He pointed out that researchers are using primary cells more and more, rather than a particular cell type.
“As soon as you need to transfect DNA, siRNA, or microRNAs into a primary cell, a lot of the conventional methods do not work as well,” said Maker. “We want to make sure that as people are beginning to transition over to using primary cells, particularly hard-to-transfect cell lines, they are able to do so using Nucleofector technology.”
In July, Amaxa announced that it will coordinate a European collaboration of eight partners to develop a device for ultra high-throughput delivery and screening of primary human cells using a 384-well platform (see CBAN, 7/6/07).
The 36-month project, which has received €2.8 million ($3.7 million) in funding from the European Commission, will use Nucleofector technology to develop the devices, which the partners said will be the first technology for HTS in efficiently transfected and differentiated primary cells.
“What HAIB is doing is based on the existing technology,” said Maker. “What we are trying to do with the 384-well format is create another step change in the technology, so that it is even more useful by those who want to look at whole genome screens or certain compound libraries.”