Far-red fluorescent tags for protein imaging in living tissues

Abstract

A vast colour palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolour labelling and whole-body imaging techniques. In the present paper, we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.

Xenopus TACC1 is a microtubule plus‐end tracking protein that can regulate microtubule dynamics during embryonic development

“…RNA was purified with LiCl precipitation and re‐suspended in nuclease free water. Constructs used: GFP‐TACC1, GFP‐TACC1‐Cterm, GFP‐TACC1‐Nterm, GFP‐TACC3 [TACC3 pET30a was gift from Richter lab (University of Massachusetts Medical, Worcester, MA)], mKate2‐TACC3 (all TACC constructs subcloned into pCS2+ vector), mKate2‐tubulin [Shcherbo et al, 2009] in pT7TS, EB1‐GFP in pCS107 [gift from Danilchik lab (Oregon Health Sciences University, Portland, OR)], mKate2‐EB1 in pCS2+. The dorsal blastomeres of embryos were injected four times at the two‐to‐four cell stage (in 0.1× MMR containing 5% Ficoll) with total mRNA amount per embryo: 1000–2000 pg GFP‐TACC1, 100 to 300 pg EB1‐GFP or mKate2‐EB1, 2000 pg mKate2‐tubulin, 1000–3000 pg mKate2‐TACC3.…”

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

“…To complement the colors of the vectors LeGO-G2 (expressing EGFP, green) and LeGO-V2 (expressing Venus, yellow), 17 four other fluorescent proteins were cloned: namely, EBFP2 (blue; a gift from Robert Campbell) (plasmid 14891; Addgene), 36 T-Sapphire (violet excitable green; a gift from Oliver Griesbeck), 37 mOrange2 (orange; a gift from Lalita Ramakrishnan) (plasmid 30175; Addgene), 38 and dKatushka2 (red; a gift from Lalita Ramakrishnan) (plasmid 30181; Addgene). 39 The six LeGO vectors used for OBC are shown in Table 1. OBC vectors can also be combined with other transgenes of interest.…”

A monomeric red fluorescent protein with low cytotoxicity

“…3 and Supplementary movies [1][2][3][4][5][6][7][8]. Owing to the lack of a statistical methodology to make direct comparisons of fluorescent protein performance in fusions, the practice is utilized only to provide one or several representative images that demonstrate the ability of the reporter to properly perform in each of the constructs.…”