cDNA

GFP-Myo10

135403

GFP-Myo10

Kanamycin

The official HUGO gene symbol for the myosin-X heavy chain gene is Myo10. It is important not to confuse Myo10 with the non-muscle myosin-IIb heavy chain, whose gene symbol is Myh10. Because Myo10 and Myh10 can be easily confused when names like myosin-10 , M10 , or myosin heavy chain 10 are used, we strongly recommend that Myo10 be used to unambiguously denote the Myo10 gene, plasmids, and protein. The GFP-Myo10 mammalian expression plasmid (11095 bp) was made by Jonathan S. Berg in the Richard E. Cheney lab and consists of nucleotides 221-6588 of the full-length bovine myosin-X heavy chain GenBank sequence U55042.1 inserted into the BglII and HindIII sites of the Clontech pEGFP-C2 vector. The GFP is fused to the N-terminus of the full-length Myo10 heavy chain (aa 1-2058). GFP-Myo10 is widely used and published because it localizes to the tips of filopodia, with the first published description in Berg, JS and Cheney, RE, Nature Cell Biol 4:246-250, 2002. Overexpressing full-length Myo10 induces numerous filopodia (Bohil, AB, Robertson, BW, and Cheney, RE, PNAS 103:12411-12416, 2006) and this can lead to major changes in cell morphology and behavior. The construct uses Myo10's natural stop codon and includes several hundred bp of 3'UTR between the stop codon and the natural Hindlll site. The GFP-Myo10 construct was generated via a complex cloning strategy that included a triple ligation due to an internal BglII site in the Myo10 tail. The GFP-Myo10 construct tends to give lower transfection efficiency than GFP alone, probably due to its larger size. The pEGFP-C2 backbone contains a neomycin resistance cassette, so use Kan plates for bacterial selection and G418 for eukaryotic selection. Relative to GenBank Myo10 cDNA sequence U55042.1 and its numbering, the Myo10 sequence in this plasmid differs at 3 sites (U55042.1>GFP-Myo10): (1) g408a gag>gaa (silent) (2) t509c atc>acc (I96T)--Most Myo10's have an I at the position, but a few have a T as do some other myosins. This change probably arose from PCR but does not block Myo10's ability to localize to filopodial tips or induce filopodia. (3) a2435g cag>cgg (Q738R)--The bovine genome sequence has a g at this position, so the g at this position in the plasmid appears to be correct and/or a natural polymorphism.

Expresses GFP-tagged bovine Myo10 in mammalian cells.

37

High Copy