Introduction

Cre/lox technology

Cre-mediated site-specific recombination has emerged as an indispensable tool for the precise manipulation of the mammalian genome. Cre promotes strand exchanges between loxP target sites without any additional cofactors (Dymecki and Kim,2007; Feil,2007). In addition to native Cre, chimeric Cre recombinases have been developed that allow temporal control of Cre-mediated recombination. Currently, Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreERT2) has the best properties for ligand sensitivity and inducible recombination efficiency (Metzger et al.,1995; Feil et al.1997). Recently, our lab showed that Cre is also highly efficient in developing and adult zebrafish and temporal control of recombination can be achieved by using the ligand-inducible CreERT2 (Hans et al., 2009; Hans et al. 2011, Kroehne et al. 2011).

Figure 1: Ligand-dependent Cre-mediated recombination. Scheme of the ligand-dependent recombination event in cells of the red-to-green reporter line. The chimeric CreERT2 recombinase is retained in the cytoplasm in the absence of the ligand. After administration of TAM which is converted to the active ligand 4-OHT, CreERT2 translocates to the nucleus, where it catalyzes the recombination event (Hans et al., 2009).

To verify the functionality of our novel CreERT2-driver lines we apply a standardized transactivation paradigm (Figure 2). In this paradigm the respective CreERT2-driver line is combined with the Cre-dependent reporter line Tg(hsp70l:loxP-DsRed-loxP-EGFP) which expresses DsRed2 under the control of the ubiquitous, temperature inducible hsp70l promoter, but switches permanently to EGFP after a successful recombination event (Kroehne et al. 2011). The progeny of this cross are exposed to 5µM Tamoxifen from 6 hpf to 24 hpf to elicit recombination, heat shocked at 24 hpf for 1 hour to activate reporter expression and analyzed at 28 hpf. CreERT2-driver lines with an onset of CreERT2 beyond 24 hpf are exposed to 5µM Tamoxifen from 36 hpf to 48 hpf, heat shocked at 48 hpf and analyzed at 52 hpf.

Figure 2: Transactivation paradigm

Generation of CreERT2 insertions

In order to obtain a wide variety of CreERT2 insertions a gene trap approach was chosen using a vector containing a splice acceptor and a mCherry-tagged variant of CreERT2 (consisting of a single open reading frame coding for mCherry and CreERT2 separated by the viral T2A peptide sequence) followed by a polyadenylation signal (Hans et al., 2011). When inserted into an intron and upon transcriptional activation of the trapped gene, a fusion transcript is generated in which the exons upstream of the insertion site are spliced to the mCherry-T2a-CreERT2 cassette.

Figure 3: Scheme of the mCherry-T2A-CreERT2 gene trap construct, containing a splice acceptor and a mCherry-tagged variant of CreERT2 (consisting of a single open reading frame coding for mCherry and CreERT2 separated by the viral T2A peptide sequence) followed by a polyadenylation signal (Hans et al., 2011). When inserted into an intron and upon transcriptional activation of the trapped gene, a fusion transcript is generated in which the exons upstream of the insertion site are spliced to the mCherry-T2a-CreERT2 cassette. (A) DNA Level (B) mRNA Level (C) Protein Level

To avoid any splice acceptor site-specific integration bias three different trapping vectors were generated comprising of different splice acceptor sites:

SA1: rabbit β-globin (Kawakami et.al, 2004)
SA2: zebrafish Bcl2 (Tian, 2009)
SA3: zebrafish Gata6 (unpublished)

Using Tol2-mediated transgenesis the various trapping vectors were injected into one-cell staged embryos. The injected animals (F0) were raised to adulthood, crossed with non-injected wild-type fish and the resulting embryos (F1) were screened for mCherry expression under a fluorescent microscope. In total, 1479 fish were screened (SA1: 1034 fish; SA2: 177 fish; SA3: 268 fish) yielding similar trapping rates.

Embryos with detectable mCherry expression were kept and further analyzed (e.g. in situ hybridization to determine the expression profile, 5'RACE to identify the trapped genes). Taken together, a variety of mCherry expression patterns were observed in transgenic F1 embryos indicating that the mCherry-T2a-CreERT2 cassette was inserted in various loci in the genome and is expressed under the control of various endogenous promoters.

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