HuGE, a novel GFP-actin-expressing mouse line for studying cytoskeletal dynamics
Introduction
The actin cytoskeleton is a highly dynamic structure employed by the cell to regulate shape changes. Apart from cell crawling actin dynamics is important in endocytosis (Yarar et al., 2005), cell division (Gerisch and Weber, 2000), and secretion (Trifaro et al., 2002). Even chromosome segregation (Lenart et al., 2005), chromatin remodeling, transcription (Bettinger et al., 2004) and nuclear stability (Bohnsack et al., 2006) depend on actin.
These different processes depend on changes in length and alignment of actin filaments, parameters which are regulated by a plethora of actin-binding proteins. Hence, observing actin dynamics in live cells and tissues has become an essential technique in cell biology. GFP-tagged versions of actin are widely used to visualize the actin cytoskeleton in live cells (Choidas et al., 1998; Westphal et al., 1997). Normally, expression vectors are transfected or the respective mRNA is injected into the cell of interest. However, in tissues or whole animals this approach is limited. An alternative method is the use of transgenic mouse models expressing the GFP-actin gene under the control of a tissue-specific promoter. Such mouse lines have been described expressing GFP-actin in keratinocytes (Vaezi et al., 2002) and neurons (Fischer et al., 2000). However, a “universal GFP-actin mouse” expressing GFP-actin in a broad range of cell types and tissues has not been available yet. Our aim was to generate a mouse line by a knock-in of the GFP-actin fusion into the profilin 1 locus, placing it under the control of the authentic profilin 1 promoter. Here we demonstrate that this mouse line is suitable for studying the actin cytoskeleton in embryonic development, as well as in a wide range of organs and primary cell lines.
Section snippets
Cloning of the targeting construct and generation of huGE mice
The GFP cDNA was fused to the 5′ end of the human β-actin cDNA (Ponte et al., 1984) and the bovine growth hormone polyA signal was added at the 3′ end. The GFP-β-actin-polyA fusion gene was then cloned into the NcoI site comprising the start codon of the mouse profilin 1 gene. Downstream of the GFP-actin gene a loxP site-flanked neor cassette was introduced to allow selection of transfected embryonic stem (ES) cells. ES clones carrying a targeted knock-in allele were identified by Southern
Generation of the GFP-actin-expressing mouse line huGE
The rational for using a knock-in strategy into the profilin 1 locus instead of a regular transgene was to preserve profilin 1-like spatial and temporal control of GFP-actin expression and to avoid problems associated with multicopy integration of the expression vector. The advantage of using the profilin 1 gene is the broad expression pattern throughout embryonic development and in tissues (Witke et al., 2001). Skeletal muscle is a tissue where profilin 1 is not expressed in appreciable
Discussion
We introduce a novel GFP-actin mouse line – huGE – and characterize the expression pattern and the functionality of the GFP-actin fusion protein. The broad expression of GFP-actin during embryonic development and in a wide range of tissues and cell types makes the huGE line a versatile tool to study actin-dependent processes in the mouse.
GFP-tagging of molecules in combination with live-cell imaging has become essential to follow cytoskeletal dynamics during cell polarization, chemotaxis, and
Acknowledgement
We are grateful to Dr. P. Gunning for the human β-actin cDNA, and Dr. J. Faix for the anti-GFP antibody, and to J. Kelly-Barrett, J. Gonzalez, P. Giallonardo, and L. Tatangelo for technical help with embryo handling and ES cell injections.
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