Location: Children's Nutrition Research CenterTitle: Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle-alpha-actin gene Author
Submitted to: Genesis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/13/2010
Publication Date: 7/1/2010
Citation: Armstrong, J.J., Larina, I.V., Dickinson, M.E., Zimmer, W.E., Hirschi, K.K. 2010. Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle-alpha-actin gene. Genesis. 48:457-463. Interpretive Summary: Within each cell there is scaffolding made out of protein which helps to organize the inner components of that cell, called the cytoskeleton. One of the cytoskeletal proteins is smooth muscle alpha actin (called SMA from now on). This paper describes the initial generation of mice that express a cherry-red colored fluorescent protein (from now on called mCherry) that is localized to the cell’s membrane, and is driven by the DNA sequence of SMA. The paper describes how this was done, and explains the steps taken afterwards to verify that only the SMA protein was targeted, and nothing else in the mouse’s DNA genome was affected (by comparing these mice to others which did not have their DNA disrupted). These newly created mice are able to have their SMA expressing cells isolated from every other cell type in their body (a useful tool in scientific research), and that this marker can be combined with other fluorescent reporters, and it can be used for live imaging of the heart as the embryo is developing. Therefore, these transgenic mice will be useful for isolating live SMA-expressing cells and for studying the emergence, behavior, and regulation of SMA-expressing cells throughout their development both before and after they are born.
Technical Abstract: Smooth muscle a actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells(vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a membrane localized cherry red fluorescent protein(mCherry), driven by the full-length SMA promoter and intronic sequences. We determined that the founders and F1 progeny of five independent lines contain 1–3 copies of the mCherry-substituted BAC vector. Furthermore,we characterized the expression of SMA-mCherry in relation to endogenous SMA in the embryo and in adult tissues, and found that the transgenic reporter in each line recapitulated endogenous SMA expression at all time points. We were also able to isolate SMA expressing cells from embryonic tissues using fluorescence- activated cell sorting (FACS). We demonstrated that this marker can be combined with other vital fluorescent reporters and it can be used for live imaging of embryonic cardiodynamics. Therefore, these transgenic mice will be useful for isolating live SMA- expressing cells via FACS and for studying the emergence,behavior,and regulation of SMA-expressing cells, including vascular smooth muscle cells and pericytes throughout embryonic and postnatal development.