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The P{Raeppli-NLS} construct is a multicolor live imaging lineage-tracing tool (named after the colorful confetti used at the carnival in Basel). It allows whole-tissue labelling such that the descendents of the majority of cells in a single organ are labelled, and can be followed simultaneously relative to one another. It contains the sequences necessary for marking cells with one of four possible fluorescent proteins: E2-Orange, mKate2, mTFP1 and TagBFP. Each fluorescent protein is tagged with a nuclear localisation sequence (Tag:NLS(Unk)). The coding sequence for each fluorescent protein is downstream of an identical attP site that may be recombined to the single attB site of the construct using phiC31:int integrase. This single attB site is placed as far as possible upstream from the attP sites to minimise any bias in fluorescent protein selection. The expression of the selected fluorescent protein can be driven by either lexAop and/or UAS regulatory sequences. These regulatory sequences are flanked by loxP and lox2272 sites respectively, meaning that P1cre recombinase can be used to excise one of the enhancers in a mutually exclusive manner if desired.

Two steps are required in order for one of the four fluorescent proteins to be selected and expressed from the starting P{Raeppli-NLS} construct. First, phiC31:int expression must be induced from sequences present in the construct. In the starting P{Raeppli-NLS} construct a full Hsp70 promoter is separated from the phiC31:int coding region by a FRT cassette containing a stop codon. To activate phiC31:int expression, this cassette must be excised using a source of FLP recombinase, after which the phiC31:int coding region is expressed either under the control of the Hsp70 promoter, or under the control of the UAS regulatory sequences present in the construct (which are further upstream of the phiC31:int coding region than the Hsp70 promoter sequences). phiC31:int promotes site-specific recombination between the upstream attB site and one of the four attP sites. Recombination excises the intervening region, bringing one of the four fluorescent proteins under the control of the lexAop and/or UAS regulatory sequences (only the fluorescent protein brought closest to the regulatory sequences is expressed as a stop codon is present after each fluorescent marker coding region). In all cases, the phiC31:int coding region is excised during the recombination event, lowering potential toxicity to the genome, and making the colour choice irreversible (in addition to the irreversibility of the attB attP recombination event). In addition a mini-w marker is also always excised, acting as a useful internal control. The end product is a construct that expresses a single fluorescent protein under the control of either the lexAop and/or UAS regulatory sequences (depending on the drivers and whether P1cre has been used to excise one of these enhancers).

Activation of P{Raeppli-NLS} in the primordial cells of a tissue results in expression of one of the four fluorescent proteins in the primordial cells. These cells divide to form clones that are stably marked by the colour of the chosen fluorescent protein, producing clones of four different colours. Activating two copies of P{Raeppli-NLS} results in ten possible combinatorial colours. This labelling can thus be used to trace the lineage of cells in a developing tissue relative to each other.

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