This article has been updated from a previous version, which incorrectly identified the company as Axxam Pharmaceuticals.
Milan, Italy-based Axxam continues to develop and refine its proprietary HTS luminescent protein, Photina, with the goal of creating a broad range of primary cells expressing the reporter for use in high-throughput screening.
Sabrina Corazza, head of the enabling technologies department at Axxam, explained that Photina is a chimeric photoprotein that can detect any variation in intracellular Ca2+ concentration. At last month’s Society for Biomolecular Sciences meeting in Montreal, Corazza discussed a project underway at the firm to generate murine embryonic stem cells that can stably express Photina at physiological levels.
“From a scientific point of view, we will continue to characterize these mESCs and identify possible uses for them,” Corazza told CBA News in a follow-up interview this week. “We are still conducting research and developing protocols.”
Ultimately, however, Corazza said that Axxam plans to use the Photina-expressing cell lines to screen compounds for pharma companies as part of its drug discovery services offering.
In her talk at SBS, Corazza said that the company divided the development project into two parts: one to generate the Photina-expression mESCs and another to create a transgenic mouse model, dubbed PhotoTopo, that expresses the Photina reporter.
The goal, she said, was to generate from different sources an unlimited number of primary cells expressing Photina for use in HTS.
The researchers induced differentiation of the mESCs into cardiomyocytes and neurons, said Corazza. She also said the mESC-derived Photina cells were differentiated directly in 96- and 384-well microtiter plates.
Corazza said that Axxam verified the differentiation by immunofluorescence markers and tested the functionality of the cardiomyocytes by injecting ligands, such as endothelin-1 and norepinephrine, which are able to activate specific endogenous receptors.
The investigators also obtained primary-like cells from a mouse embryonic carcinoma cell line called mEC P19 Photina, that expressed Photina. Corazza pointed out that mEC cells are easier to cultivate than mESC cells and do not require feeder cells to support growth and maintain an undifferentiated state.
In addition, Corazza said it has been well-documented in the literature that these cells can efficiently differentiate in vitro into neurons and cardiomyocytes. Corazza said that the Axxam team induced the differentiation of the P19 Photina cells into cardiomyocytes and neurons directly in 96- and 384-well microtiter plates, as they did with the mESCs.
As was done with the mESCs, the functionality of the differentiated mEC-derived neurons was also verified by the addition of ligands for specific endogenous receptors.
That’s Italian for Mouse
“We also decided to generate transgenic mice expressing Photina,” Corazza said. She explained that the Axxam researchers call these mice PhotoTopo, because the animals express the Photina reporter, and topo is Italian for mouse.
“The transgenic mice and the reporter mESCs have potential for use in many different applications.”
“To generate the PhotoTopo mice, we injected mESCs that expressed Photina into the blastocyst and then crossed the resulting chimeric mice with a wild-type C57/BL6 mouse,” Corazza said.
The investigators first obtained heterozygous F1 mice and then possibly homozygous and heterozygous F2 progeny expressing Photina, said Corazza. She said that the Photina reporter was present in most of the tissue samples isolated from the heterozygous progeny. She also said that the researchers found that the level of Photina expression was kept constant in mice from newborns up to eight months.
The Axxam team was able to isolate different types of primary cells from the PhotoTopo mice, Corazza said, including macrophages derived from femoral bone marrow and pancreatic beta islet cells. “In all of these cells, we were able to detect the presence of the reporter gene, and we used these cells in 96- and 384-well microtiter plates for cell function analysis,” she said.
Isolated primary cells from PhotoTopo mice are a source of cells expressing a calcium-activated reporter gene for HTS or a source of adult stem cells expressing a calcium-activated reporter gene, said Corazza. The PhotoTopo mice can be also be used for photoimaging studies and crossed with pharmacologically relevant mutant mice.
“The transgenic mice and the reporter mESCs have potential for use in many different applications,” Corazza concluded.