fos promoter  (Addgene inc)

Journal: Nature biotechnology

Article Title: Time-tagged ticker tapes for intracellular recordings.

doi: 10.1038/s41587-022-01524-7

Figure Lengend Snippet: Fig. 1 | iPAK4 forms intracellular protein fibers. a, Scheme for intracellular recording of cFos activity with fiducial timestamps. iPAK4 forms the fiber scaffold. HaloTag-iPAK4 incorporates HT dyes, permitting labeling of the fiber with fiducial timestamps. Neural activation drives expression of cFos::eGFP- iPAK4, introducing green bands into the fiber. b, Composition of the protein constructs used to label intracellular protein fibers. c, The structures of tagged iPAK4 monomers and the crystal structure with hexagonal pores (from the Protein Data Bank: 4XBR) were modeled using Protein Imager software35. d, Image of a HEK cell expressing CMV::iPAK4 (95%) and CMV::HT-iPAK4 (5%). The fiber was stained with JFX608; the membrane was labeled by expressing

Article Snippet: All iPAK4 constructs were cloned into a second-generation lentiviral backbone (Addgene, 136636) with either a cytomegalovirus (CMV) or a cFos promoter (Addgene, 47907) using standard Gibson assembly36.

Techniques: Activity Assay, Labeling, Activation Assay, Expressing, Construct, Staining, Membrane

Journal: Nature biotechnology

Article Title: Time-tagged ticker tapes for intracellular recordings.

doi: 10.1038/s41587-022-01524-7

Figure Lengend Snippet: Fig. 5 | Protein ticker tape recordings of cFos activation in neurons. a, Image of a cultured neuron expressing lentiviral CMV::iPAK4 (90%) and CMV::eGFP- iPAK4 (10%). Scale bar, 50 μm. b, Representative patch-clamp recordings in neurons with or without an iPAK4 fiber. Spikes were evoked by a current injection of 100 pA. c, There were no significant differences between neurons with or without fibers in membrane resistance (404 ± 73 MΩ versus 339 ± 43 MΩ, P = 0.43), membrane capacitance (63 ± 6 pF versus 67 ± 9 pF, P = 0.67), resting potential (−61.2 ± 1.2 mV versus −60.2 ± 0.8 mV, P = 0.48) or rheobase (89 ± 12 pA versus 109 ± 13 pA, P = 0.27) (n = 11 neurons with fibers, 12 neurons without). Error bars show mean ± s.e.m. Two-sided Student’s t-test was employed for data comparison. d, Fiber in a neuron expressing lentiviral CMV::iPAK4 (90%) and cFos::eGFP-iPAK4 (10%). The top image shows the fiber after a first PMA addition;

Article Snippet: All iPAK4 constructs were cloned into a second-generation lentiviral backbone (Addgene, 136636) with either a cytomegalovirus (CMV) or a cFos promoter (Addgene, 47907) using standard Gibson assembly36.

Techniques: Activation Assay, Cell Culture, Expressing, Patch Clamp, Injection, Membrane, Comparison