pcag ert2 creert2  (Addgene inc)

Journal: Disease Models & Mechanisms

Article Title: Fate mapping melanoma persister cells through regression and into recurrent disease in adult zebrafish

doi: 10.1242/dmm.049566

Figure Lengend Snippet: A tamoxifen-inducible mitfa:cre ERt2 /loxP system in zebrafish labels melanocytes. (A) A schematic of mitfa:cre ERt2 (left) and ubi:Switch (right) constructs to enable a green-to-red switch in mitfa promoter-expressing cells after tamoxifen treatment. The cardiac myosin light chain 2 ( cmlc2 ; also referred to as myl7 ) promoter drives GFP expression and facilitates screening for the cre ERt2 line based on GFP + heart myocardium. mCh, mCherry. (B) A schematic of the concept behind the colour-switching cre ERt2 /loxP system that allows green-to-red switch after tamoxifen (T) treatment in cells expressing the mitfa promoter driving cre ERt2 . (Left) A three-night course of treatment with tamoxifen for 11-12 h by immersion in the dark with a drug-free period during the day. (Right) The principle of colour switching upon tamoxifen treatment as a result of Cre ERt2 trans-localisation to the nucleus, causing excision of GFP and a stop codon, enabling mCherry expression in recombined cells. (C) An illustration of the expected green-to-red fluorophore switch in the zebrafish embryo upon three consecutive daily treatments with 20 µM 4-hydroxytamoxifen (4-OHT). Enlarged view of dashed line rectangle shows ‘switched’ melanocytes in the embryonic stripes. hpf, h post fertilisation. (D,E) Lateral (D) and dorsal (E) views of a zebrafish larva after 4-OHT treatment at 104 hpf (4.5 dpf) validates mitfa:cre ERt2 specificity to melanocyte lineage. Standard deviation intensity (STD) projection of a confocal z -stack (left, mCherry) and merged with a single z -plane acquisition in brightfield (BF) channel (right, merge) at 6 h after the end of 4-OHT treatment. mCherry-expressing melanocytes are visible and express black melanin pigment (pink arrows). Two unpigmented, star shaped cells expressing mCherry are also visible and may represent xanthophore progenitors (yellow arrows). N =6 fish for each view, scale bars: 100 µm.

Article Snippet: Cre ERt2 was amplified by PCR using pCGA_cre ERt2 (Addgene plasmid # 14797 ) as template.

Techniques: Construct, Expressing, Standard Deviation

Journal: Disease Models & Mechanisms

Article Title: Fate mapping melanoma persister cells through regression and into recurrent disease in adult zebrafish

doi: 10.1242/dmm.049566

Figure Lengend Snippet: Successful fluorophore switch using the zebrabow system in adult zebrafish primary melanoma following tamoxifen treatment. (A) A schematic of mitfa:cre ERt2 and ubi:zebrabow constructs , which enables recombination from red signal (RFP) to a stochastic combination of cyan (CFP), yellow (YFP) and red (RFP) signal in mitfa:cre ERt2 -expressing cells after tamoxifen (T) treatment. The cmlc2 promoter drives GFP expression and facilitates screening for the cre ERt2 line based on GFP + heart myocardium. (B) Multicolour labelling in adult zebrafish melanomas. An adult zebrafish with two primary melanomas 8 days before the tamoxifen treatment course (top), and 6 days (middle) and 34 days (bottom) after the start of the treatment. Increasing de novo expression of CFP and YFP proteins in both pigmented and unpigmented tumours can be detected after tamoxifen treatments (4 µM). N =3 fish, scale bars: 1 mm, white arrows point to two tumour locations. The arrows on the brightfield image (Day 34) point to the melanomas shown in panels C and E, respectively. (C) Confocal multicolour imaging of zebrafish melanoma. Single z -plane of confocal acquisition of tamoxifen-treated fish shows individual cells acquiring varied combinations of CFP, YFP and RFP (blue, green and yellow arrows on merged image, respectively). N =3 fish, scale bar: 100 µm. (D) Overview of the vibratome sectioning protocol. The PFA-fixed tissue is mounted in 4% agarose and cut using a vibrating blade into 400 µm-thick sections to capture the pigmented tumour (E). (E) Vibratome section imaging of a tamoxifen-treated fish (tissue location as shown in B). Clusters of CFP, YFP or double-expressing cells are clearly visible in areas of the pigmented tumour. STD projections of confocal z -stacks of a PFA-fixed sectioned tissue. N =3 fish, scale bars: 200 µm. See also Fig. S1B-D .

Article Snippet: Cre ERt2 was amplified by PCR using pCGA_cre ERt2 (Addgene plasmid # 14797 ) as template.

Techniques: Construct, Expressing, Imaging