Known as the GeneSwap approach, this new technology provides novel insights into mitochondrial diseases, which are often lethal and have only palliative treatments available.
By Lindsay Lyle
Scientists at the Frederick P. Whiddon College of Medicine at the University of South Alabama have developed a technique to perform reverse genetic analysis of proteins involved in mitochondrial DNA (mtDNA) replication. Known as the GeneSwap approach, this new technology provides novel insights into how defects in mtDNA replication cause mitochondrial diseases, which are often lethal and have only palliative treatments available.
Mikhail Alexeyev, Ph.D., professor of physiology and cell biology, is the senior author of a study recently published in Cells, an international, peer-reviewed journal of cell biology, molecular biology and biophysics.
The lack of approaches for reverse genetic analysis of mtDNA replication presented a major obstacle to better understanding mtDNA replication and, consequently, developing effective treatment strategies for disorders caused by defects in this process, Alexeyev said.
“Only a person who knows how the car operates can fix it,” he explained. “In the same way, knowing how a disease develops, its mechanism, allows us to intervene and disrupt it.”
Classical, or “forward,” genetics starts with a trait and works to identify a responsible gene or an alteration in a gene. Reverse genetics, on the other hand, is a relatively recent development made possible by advances in molecular biology and genetic engineering.
“It works in the opposite direction: We alter, or mutate, a gene in a test tube and examine changes in this gene's or its product's function,” Alexeyev said. “The advantage of reverse genetics is that it allows the generation and testing of specific hypotheses as to how a given gene or its product may work.”
The lab utilized genetic recombination systems encoded by bacterial viruses (bacteriophages) and mutant genes carried by a type of disabled mouse virus to substitute, in one step, a mutant gene for a normal one in cultured cells.
Using the GeneSwap approach, Alexeyev and the research team identified 730 conditionally permissive mutations in mitochondrial transcription factor A (TFAM), compared to fewer than a dozen that were known prior to their study. They also made several other seminal discoveries that will be published in upcoming papers.
Additional authors of the study are Natalya Kozhukhar, research technologist; Domenic Spadafora, Ph.D.; Flow Cytometry Core Lab manager; and Yelitza A. R. Rodriguez, Ph.D., a recent graduate in basic medical sciences.
Read the full article in Cells: “A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication.”