mTagBFP2 fluorescent protein expression vectors were constructed using -C1 and -N1 (Clontech-style) cloning vectors. The mTagBFP2 cDNA was amplified with a 5′ primer encoding an AgeI site and a 3′ primer encoding either a BspEI (-C1) or NotI (-N1) site for C-terminal and N-terminal fusions (with regards to the FP), respectively. Purified and digested PCR products were ligated into similarly digested pEGFP-C1 and pEGFP-N1 cloning vector backbones. To generate targeting fusion vectors, the appropriate cloning vector and a previously assembled EGFP fusion vector were digested, either sequentially or doubly, with the appropriate enzymes and ligated together after gel purification.
Thus, to prepare mTagBFP2 C-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human lamin B1 (10), NheI and BglII (lamin B1 cDNA source: George Patterson, NIH; NM_005573.2); 20 amino acid farnesylation signal from c-Ha-Ras (CAAX; 5), AgeI and BspEI (c-Ha-Ras cDNA source: Clontech, Mountain View, CA; NM_001130442.1); endoplasmic reticulum (5), AgeI and BspEI (calreticulin cDNA source: George Patterson, NIH; NM_004343.3); fibrillarin (7), AgeI and BspEI (fibrillarin cDNA source: Evrogen, Moscow, Russia; NM_001436.3); human light chain clathrin (15), NheI and BglII (clathrin cDNA source: George Patterson, NIH; NM_001834.2); β-actin (7), NheI and BglII (human β-actin cDNA source: Clontech, Mountain View, CA; NM_001101.3); caveolin 1 (10), NheI and BglII (human caveolin 1 cDNA source: Origene, Rockville, MD; NM_001753); vinculin (22) AgeI and EcoRI (human vinculin source: Clare Waterman, NIH; NM_003373.3); CAF1 (10), AgeI and BspEI (mouse chromatin assembly factor cDNA source: Akash Gunjan, FSU; NM_013733.3) Rab5a (7), NheI and BglII (canine Rab5a cDNA source: Vicki Allen, University of Manchester; NM_001003317.1); α-tubulin (18), NheI and BglII (human α-tubulin cDNA source: Clontech, Mountain View, CA; NM_006082); myosin IIA (18) NheI and BglII (human myosin heavy chain IIA cDNA source: DNA2.0, Menlo Park, CA; AJ312390.1); PCNA (19), AgeI and BspEI (proliferating cell nuclear antigen cDNA source: David Gilbert, FSU; NM_002592.2).
To prepare mTagBFP2 N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: β-2 connexin-26 (7), BamHI and NotI (rat Cx26 cDNA source: Matthias Falk, Lehigh U; NM_001004099.1); TfR (20), BamHI and NotI (transferrin receptor cDNA source: George Patterson, NIH; NM_NM_003234); Golgi complex (7), BamHI and NotI (human β-galactosamide α-2,6-sialyltransferase 1cDNA source: Jennifer Lippincott-Schwartz, NIH; NM_173216.2); zyxin (6), BamHI and NotI (human zyxin cDNA source: Origene, Rockville, MD; NM_003461); vascular epithelial cadherin (10), AgeI and NotI (human VE cadherin cDNA source: Origene, Rockville, MD; NM_001795.3); mitochondria (7), BamHI and NotI (human mitochondrial targeting sequence, cytochrome c oxidase cDNA source: Clontech, Mountain View, CA; NM_004074.2); centromere protein B (22), BamHI and NotI (human CENPB cDNA source: Alexey Khodjakov, Wadsworth Center, Albany, NY; NM_001810.5); α-actinin (19), BamHI and EcoRI (human α-actinin cDNA source: Tom Keller, Florida State University, Tallahassee; NM_001130005.1); c-src sarcoma (7), BamHI and EcoRI (chicken c-src cDNA source: Marilyn Resh, Sloan-Kettering, New York; XM_001232484.1); Lifeact (7), BamHI and NotI (Lifeact cDNA source: IDT, Coralville, IA); vimentin (7), BamHI and NotI (human vimentin cDNA source: Robert Goldman, Northwestern University; NM_003380.3).
All DNA for transfection was prepared using the Plasmid Maxi kit (QIAGEN, Valencia, CA). To ensure proper localization, mTagBFP2 fusion proteins were characterized by transfection in HeLa cells (CCL2 line; ATCC, Manassas, VA) using Effectene (QIAGEN) and 1 µg vector. Transfected cells were grown on coverslips in DMEM/F12, fixed after 48 hours, and mounted with Gelvatol. Epifluorescence images (Figure 4 ) were taken with a Nikon 80i microscope using widefield illumination and an Omega QMax Blue filter set to confirm proper localization.
Thus, to prepare mTagBFP2 C-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human lamin B1 (10), NheI and BglII (lamin B1 cDNA source: George Patterson, NIH; NM_005573.2); 20 amino acid farnesylation signal from c-Ha-Ras (CAAX; 5), AgeI and BspEI (c-Ha-Ras cDNA source: Clontech, Mountain View, CA; NM_001130442.1); endoplasmic reticulum (5), AgeI and BspEI (calreticulin cDNA source: George Patterson, NIH; NM_004343.3); fibrillarin (7), AgeI and BspEI (fibrillarin cDNA source: Evrogen, Moscow, Russia; NM_001436.3); human light chain clathrin (15), NheI and BglII (clathrin cDNA source: George Patterson, NIH; NM_001834.2); β-actin (7), NheI and BglII (human β-actin cDNA source: Clontech, Mountain View, CA; NM_001101.3); caveolin 1 (10), NheI and BglII (human caveolin 1 cDNA source: Origene, Rockville, MD; NM_001753); vinculin (22) AgeI and EcoRI (human vinculin source: Clare Waterman, NIH; NM_003373.3); CAF1 (10), AgeI and BspEI (mouse chromatin assembly factor cDNA source: Akash Gunjan, FSU; NM_013733.3) Rab5a (7), NheI and BglII (canine Rab5a cDNA source: Vicki Allen, University of Manchester; NM_001003317.1); α-tubulin (18), NheI and BglII (human α-tubulin cDNA source: Clontech, Mountain View, CA; NM_006082); myosin IIA (18) NheI and BglII (human myosin heavy chain IIA cDNA source: DNA2.0, Menlo Park, CA; AJ312390.1); PCNA (19), AgeI and BspEI (proliferating cell nuclear antigen cDNA source: David Gilbert, FSU; NM_002592.2).
To prepare mTagBFP2 N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: β-2 connexin-26 (7), BamHI and NotI (rat Cx26 cDNA source: Matthias Falk, Lehigh U; NM_001004099.1); TfR (20), BamHI and NotI (transferrin receptor cDNA source: George Patterson, NIH; NM_NM_003234); Golgi complex (7), BamHI and NotI (human β-galactosamide α-2,6-sialyltransferase 1cDNA source: Jennifer Lippincott-Schwartz, NIH; NM_173216.2); zyxin (6), BamHI and NotI (human zyxin cDNA source: Origene, Rockville, MD; NM_003461); vascular epithelial cadherin (10), AgeI and NotI (human VE cadherin cDNA source: Origene, Rockville, MD; NM_001795.3); mitochondria (7), BamHI and NotI (human mitochondrial targeting sequence, cytochrome c oxidase cDNA source: Clontech, Mountain View, CA; NM_004074.2); centromere protein B (22), BamHI and NotI (human CENPB cDNA source: Alexey Khodjakov, Wadsworth Center, Albany, NY; NM_001810.5); α-actinin (19), BamHI and EcoRI (human α-actinin cDNA source: Tom Keller, Florida State University, Tallahassee; NM_001130005.1); c-src sarcoma (7), BamHI and EcoRI (chicken c-src cDNA source: Marilyn Resh, Sloan-Kettering, New York; XM_001232484.1); Lifeact (7), BamHI and NotI (Lifeact cDNA source: IDT, Coralville, IA); vimentin (7), BamHI and NotI (human vimentin cDNA source: Robert Goldman, Northwestern University; NM_003380.3).
All DNA for transfection was prepared using the Plasmid Maxi kit (QIAGEN, Valencia, CA). To ensure proper localization, mTagBFP2 fusion proteins were characterized by transfection in HeLa cells (CCL2 line; ATCC, Manassas, VA) using Effectene (QIAGEN) and 1 µg vector. Transfected cells were grown on coverslips in DMEM/F12, fixed after 48 hours, and mounted with Gelvatol. Epifluorescence images (
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