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- EXPERIMENTAL PROCEDURES
Cre-mediated site-specific recombination has become an invaluable tool for manipulation of the murine genome. The ability to conditionally activate gene expression or to generate chromosomal alterations with this same tool would greatly enhance zebrafish genetics. This study demonstrates that the HSP70 promoter can be used to inducibly control expression of an enhanced green fluorescent protein (EGFP) –Cre fusion protein. The EGFP–Cre fusion protein is capable of promoting recombination between lox sites in injected plasmids or in stably inherited transgenes as early as 2 hr post–heat shock induction. Finally, the levels of Cre expression achieved in a transgenic fish line carrying the HSP70-EGFP–cre transgene are compatible with viability and both male and female transgenic fish are fertile subsequent to induction of EGFP–Cre expression. Hence, our data suggests that Cre-mediated recombination is a viable means of manipulating gene expression in zebrafish. Developmental Dynamics 233:1366–1377, 2005. © 2005 Wiley-Liss, Inc.
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- EXPERIMENTAL PROCEDURES
To generate an inducible system for expressing Cre recombinase, an EGFP–cre fusion gene (Le et al., 1999b) was placed under the control of a zebrafish heat shock–inducible HSP70 promoter (Fig. 1A). Earlier studies have shown efficient transient induction of GFP using this HSP70 promoter (Halloran et al., 2000). In addition, laser induction allowed control of expression to be restricted to single cells, while subjecting an entire embryo or fish to heat shock resulted in global induction of expression. Hence, by analogy, the HSP70-EGFP–cre transgene should allow Cre expression to be controlled in specific cells or, alternatively, globally throughout the developing or adult zebrafish.
Figure 1. Cartoon of HSP70-EGFP-cre and recombination reporter plasmids. A: HSP70-EGFP-cre construct. B: CMV driven hcRFP reporter construct. Gray triangles denote lox511 sites, and the red stop sign denotes the synthetic polyadenylation site and C2 termination site. C: PGK promoter driven lacZ reporter construct. Gray triangles denote loxP sites, and the red stop sign denotes the insertion of the neomycin (or G418) resistance gene and SV40 polyadenylation site. D: CMV-driven EGFP reporter (Cre Stoplight) construct. Gray triangles denote loxH (immediately 3′ to the CMV promoter) and loxP (3′ to the stop sign) sites, and the red stop sign denotes the insertion of the dsRFP gene and bovine growth hormone gene polyadenylation site. B–D: Small bent arrows denote transcriptional start sites, and the EGFP–Cre fusion protein is denoted by the fused green and blue spheres.
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The original HSP70-EGFP construct was injected into one-cell embryos to generate mosaic transgenic zebrafish. These mosaic transgenic embryos at 24 hr postfertilization (hpf) were heat shock–induced for 90 sec at 42°C. Confirming the results of an earlier study (Halloran et al., 2000), no EGFP expression was seen in the absence of heat shock (Fig. 2A), but heat shock induction caused strong EGFP expression in a subset of cells (Fig. 2B). To test initially the Cre system, mosaic transgenic embryos were generated by injection of the HSP70-EGFP-cre vector into one-cell embryos. As expected, no EGFP-Cre expression was detected in the absence of heat shock (Fig. 2C). In contrast, strong mosaic EGFP-Cre expression (at comparable levels to the EGFP expression generated using the HSP70-EGFP vector) was seen subsequent to 90 sec of heat shock (compare Fig. 2D with 2B). Notably, initiation of EGFP-Cre expression was evident as early as 2 hr, appeared to achieve peak levels of expression by 4–6 hr and was still evident 24 hr post–heat shock induction.
Figure 2. Heat shock regulated expression of EGFP and EGFP–Cre. A: Mosaic transgenic zebrafish (24 hours postfertilization [hpf]) for the HSP70-EGFP construct in the absence of heat shock. B: Mosaic transgenic zebrafish (24 hpf) for the HSP70-EGFP construct heated for 90 sec at 42°C. C: Mosaic transgenic zebrafish (24 hpf) for the HSP70-EGFP-cre construct in the absence of heat shock. D: Mosaic transgenic zebrafish (24 hpf) for the HSP70-EGFP-cre construct heated for 90 sec at 42°C. E: Confocal Z-stack showing a representative HSP70-EGFP-cre Tg/Tg zebrafish at approximately 24 hpf. Brightfield images of the embryos in A–D are shown in the insets. Scale bars = 250 microns in B (applies to inset and A–D), 500 microns in E.
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Some of the mosaic HSP70-EGFP-cre transgenic fish were raised to sexual maturity. The offspring of these fish were examined for germline transmission of the transgene by heat induction for 90 sec at 42°C followed by screening under fluorescence imaging. Approximately 15% (84 of 575) of the progeny of one of the mosaic founders carried the transgene and demonstrated uniform expression of EGFP-Cre (Fig. 2E), whereas only 0.5% (3/660) of the progeny from a second founder carried the transgene (data not shown). Importantly, the frequency of long-term survival of fish carrying the EGFP-cre transgene subsequent to heat shock induction of EGFP-Cre was no different than wild-type hatch mates (Table 1). Both male and female transgenic fish were fertile. Furthermore, an intercross of fish carrying one copy of the transgene (Tg/+) yielded Mendelian ratios of Tg/Tg, Tg/+, and +/+ offspring (N = 27, 52, and 21, respectively). In this cross, the Tg/Tg offspring were distinguished from the Tg/+ offspring by the increased intensity and earlier uniform detection of EGFP fluorescence after heat shock induction (data not shown). Again, no increased lethality was observed in the heat shock–induced Tg/+ and Tg/Tg offspring.
Table 1. Survival of Offspring Following Heat Shock Induction at 42°C for 90 sec at 24 hpfa
|Age that fish were analyzed||Tg-positive||Tg-negative|
|1 day||100% (N = 90)||100% (N = 77)|
To more accurately compare heat shock induction schemes in the HSP70-EGFP-cre Tg/+ embryos, a HSP70-EGFP-cre Tg/Tg male was crossed to a wild-type female and the resulting Tg/+ embryos from the same clutch were either maintained at 28.5°C or heat shocked under varying conditions. No fluorescent signal above the background autofluorescence was seen at 24 hpf in the un-induced Tg/+ embryos (Fig. 3A). The edge effect on the embryo in Figure 3A was not true EGFP fluorescence, rather it was due to using exposure conditions that allow focusing on the un-induced embryo and reflection from the yolk sac. In contrast, induction for 15 min at 37°C at 20 hpf resulted in low but detectable levels of EGFP-Cre fluorescence at 24 hpf (Fig. 3B). Furthermore, when the population of embryos for the various induction schemes was compared, 30 min of induction at 37°C at 20 hpf resulted in noticeably higher levels of EGFP-Cre fluorescence than the 15-min induction (compare Fig. 3B with 3C). Slight increases in intensity of fluorescence were seen for 60 min (or 270-min induction) at 37°C starting at 20 hpf (data not shown). In addition, an increasing number of the embryos in the heat shocked population achieved the highest fluorescence level with longer induction. Finally, a 5-min induction at 42°C at 20 hpf also gave strong EGFP-Cre fluorescence in the anterior end of the embryo, but fluorescence in the body was weaker than the 30-min 37°C induction (compare Fig. 3D with 3C). As we had already carefully monitored embryo survival after a 90-sec heat shock at 42°C and found no discernible survival differences relative to wild-type embryos (Table 1), we monitored the survival of embryos that were heat shocked under these different parameters for 2 weeks post-heat shock (Table 2). Importantly, these different regimens did not negatively affect embryo survival through 2 weeks post-heat shock.
Figure 3. Analysis of heat shock induction schemes for HSP70-EGFP-cre transgenic line. A: Un-induced HSP70-EGFP-cre Tg/+ embryo photographed at 24 hours postfertilization (hpf). B: HSP70-EGFP-cre Tg/+ embryo photographed at 24 hpf after being induced for 15 min at 37°C at 20 hpf. C: HSP70-EGFP-cre Tg/+ embryo photographed at 24 hpf after being induced for 30 min at 37°C at 20 hpf. (D) HSP70-EGFP-cre Tg/+ embryo photographed at 24 hpf after being induced for 5 min at 42°C at 20 hpf.
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Table 2. Viability of HSP70-EGFP-cre Fish Following Different Heat-Shock Parameters
|Method of heat shock||Pre-heat shock||1 day post-heat shock||2 days post-heat shock||1 week post-heat shock||2 weeks post-heat shock|
|37°C, 15 min||117||116 (99%)||115 (98%)||113 (97%)||104 (89%)|
|37°C, 30 min||113||112 (99%)||112 (99%)||108 (96%)||105 (93%)|
|37°C, 60 min||127||126 (99%)||125 (98%)||125 (98%)||117 (92%)|
|37°C, 270 min||151||151 (100%)||151 (100%)||151 (100%)||132 (87%)|
|42°C, 5 min||106||102 (96%)||102 (96%)||102 (96%)||93 (88%)|
The first stable HSP70-EGFP-cre transgenic line was examined by several means. First, Southern transfer analysis was used to verify integration of the transgene. As shown in Figure 4A, restriction fragments of the expected sizes were detected in the transgenic line, but not in wild-type fish, demonstrating that the line carries one copy of the transgene. Specifically, both HindIII–AflII (Fig. 4A) and BglII–AflII (data not shown) double digestions yielded the expected specific single band of 3.6 kb. Therefore, the transgene was intact from the BglII or HindIII sites in the polylinker upstream of the heat shock promoter through the AflII site following the SV40 polyadenylation site. There were no other useful sites 5′ to the HSP70 promoter, but the presence of a 4.5-kb hybridizing fragment upon double digestion with HindIII and StuI (Fig. 4A) or BglII and StuI (data not shown) revealed that the transgene was intact for another 0.9 kb 3′ to the SV40 polyadenylation site. Second, reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to verify expression of the transgene after heat shock induction using primers upstream of the EGFP–cre fusion point and downstream of the cre stop codon. As shown in Figure 4B, the expected PCR product was only detected in the samples containing reverse transcriptase. Furthermore, the 1.2-kb PCR product from two independent RT-PCR reactions was subjected to DNA sequencing, and no point mutations were found within the cre gene. Hence, a wild-type fusion transcript is conditionally expressed in this transgenic line. Next, Western blot analysis was used to detect expression of the fusion protein. As shown in Figure 4C, antisera against Cre detected the expected 68-kDa fusion protein in transgenic fish subjected to heat shock induction (Fig. 4, lanes 4, 6, and 8), but not in un-induced transgenic fish (Fig. 4, lane 2), un-induced wild-type fish (Fig. 4, lane 1), or in wild-type fish subjected to any of the heat shock induction schemes (Fig. 4, lanes 3, 5, and 7). In contrast, different heat shock induction strategies resulted in the accumulation of varying amounts of EGFP–Cre fusion protein in transgenic fish. Induction for 1 hr at 39°C (lane 6) appeared to induce the most fusion protein and 1 hr at 37°C (lane 4) induced an intermediate amount. Of interest, a 5-min heat shock induction at 42°C (lane 8) gave the lowest level of fusion protein, yet (as will be shown below) an even shorter (90 sec) 42°C heat shock induction actively allowed Cre-mediated recombination in the transgenic fish. Only low levels of Cre protein are required for recombination of substrates and useful murine Cre-expressing lines have not generated detectable levels of Cre protein using this antisera (B.S., unpublished data). Hence, the 42°C induction appears to generate experimentally useful levels of EGFP-Cre protein. Thus, this transgenic line conditionally generates the EGFP–Cre fusion protein.
Figure 4. Analysis of HSP70-EGFP-cre transgenic line. A: Southern transfer analysis of HSP70-EGFP-cre transgenic line. B: Reverse transcriptase-polymerase chain reaction analysis of HSP70-EGFP-cre transgenic line. “H20” denotes no DNA control reaction. “+” and “−” denote addition and absence of reverse transcriptase, respectively. C: Western blot detection of EGFP–Cre fusion protein. Lane 1: un-induced wild-type fish; lane 2: un-induced HSP70-EGFP-cre Tg/+ fish; lane 3: wild-type fish heat shock induced for 1 hr at 37°C; lane 4: HSP70-EGFP-cre Tg/+ fish heat shock induced for 1 hr at 37°C; lane 5: wild-type fish heat shock induced for 1 hr at 39°C; lane 6; HSP70-EGFP-cre Tg/+ fish heat shock induced for 1 hr at 39°C; lane 7: wild-type fish heat shock induced for 5 min at 42°C; lane 8: HSP70-EGFP-cre Tg/+ fish heat shock induced for 5 min at 42°C.
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Modeled on the reporter systems routinely used to monitor Cre recombination in mice (Lobe et al., 1999; Mao et al., 1999, 2001; Srinivas et al., 2001), an hcRFP recombination reporter construct was generated. A CMV promoter-driven hcRFP, pHcRed1-C1 (Clontech), was modified to include two lox511 sites flanking an RNA polymerase II termination signal between the promoter and translational start site of hcRFP (see Fig. 1B). This reporter construct was transformed into Cre-expressing Escherichia coli (Sauer and Henderson, 1988) to excise the termination signal and generate the expected final recombination product containing a single lox511 site (see Fig. 1B). When this recombination plasmid was injected into single-cell embryos, 78% of the embryos yielded mosaic hcRFP expression (Fig. 5B; Table 3), demonstrating the reporter vector should successfully monitor Cre-mediated recombination in zebrafish.
Figure 5. Detection of hcRFP after heat shock induction of EGFP-Cre. A: Lack of detectable expression from the hcRFP reporter construct (depicted at the top of the panel) in mosaic transgenic embryos at 24 hours postfertilization (hpf). B: Detection of expression from final recombination product (depicted at the top of the panel) in mosaic transgenic embryos at 24 hpf. C–F: Detection of hcRFP-positive cells after heat shock induction of EGFP-Cre. C: Brightfield image showing the trunk of an HSP70-EGFP-cre embryo injected with the hcRFP reporter construct. D: EGFP fluorescent image of embryo shown in C, showing remaining EGFP intensity 24 hr post heat shock. E: hcRFP fluorescent image of embryo shown in C. Arrow denotes a cluster of hcRFP-positive cells 24 hr post heat shock. F: Artificial merge of brightfield and fluorescent images. Arrow denotes the cluster of hcRFP-positive cells shown in E. Scale bars = 250 microns in A (applies to A,B and insets).
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Table 3. Analysis of Recombination Events in hcRFP Transient Injection Studies
|Plasmid injected||Injected line||Heat shock||Heat shock at||Analyzed at||RFP− total||RFP+ total||N||% RFP+||Distribution of RFP-positive embryos by no. of positive cells per embryo|
|lox2 hcRFP||HSP70 EGFP-cre Tg/+||37°C, 30 min||5 hpf||29 hpf||6||14||20||70%||5||4||3||2||0||0||0|
|lox2 hcRFP||HSP70 EGFP-cre Tg/+||37°C, 60 min||24 hpf||48 hpf||14||23||37||62%||15||2||6||0||0||0||0|
|lox2 hcRFP||HSP70 EGFP-cre Tg/+||NONE||N/A||24 hpf||152||5||157||3%||4||1||0||0||0||0||0|
|lox2 hcRFPa||HSP70 EGFP-cre Tg/+||NONE||N/A||48 hpf||142||6||148||4%||5||1||0||0||0||0||0|
|lox hcRFP||wild-type||NONE||N/A||29 hpf||11||39||50||78%||6||5||12||6||6||3||1|
|lox hcRFPa||wild-type||NONE||N/A||48 hpf||11||39||50||78%||12||7||8||6||5||1||0|
Finally, to test the fully integrated system, the hcRFP reporter construct was injected into one-cell embryos from crosses of wild-type and HSP70-EGFP-cre (Tg/Tg) fish. In the absence of heat shock, 97% of the embryos (152 of 157) exhibited a complete absence of hcRFP-expressing cells at 24 hpf (Fig. 5A; Table 3). Twenty-four hours after heat shock induction of EGFP-Cre, however, 62% to 70% of the injected embryos contained mosaic groupings of hcRFP-expressing cells (Fig. 5C–F; Table 3). Because the distribution of any injected reporter construct is mosaic, hcRFP fluorescence was only observed in clusters of cells, ranging from only a few hcRFP-positive cells per embryo (Fig. 5C–F) to more than 20 cells per embryo (Table 3).
To further verify the ability of EGFP-Cre to catalyze recombination in the transgenic line, a second recombination reporter expressing β-galactosidase subsequent to Cre-mediated site-specific recombination was used (Fig. 1C). Injection of the recombination product, derived by transformation of Cre-expressing Escherichia coli (Sauer and Henderson, 1988), into wild-type one-cell embryos led to mosaic expression of β-galactosidase at 24 hpf (Fig. 6A). At 48 hpf, 41% of the injected embryos (N = 37) exhibited β-galactosidase–expressing cells (data not shown). Although this reporter construct is not as robust as the hcRFP reporter, it should still detect Cre activity in the EGFP-cre line. Hence, the recombination substrate was injected into one-cell embryos from a HSP70-EGFP-cre (Tg/+) × wild-type cross, and the embryos were heat shocked at 24 hpf. One day subsequent to heat shock induction, at 48 hpf, no β-galactosidase–expressing cells were visualized in the EGFP-Cre–negative embryos (N = 17 tested). However, β-galactosidase–expressing cells were visualized in 21% of the EGFP-Cre–positive embryos (N = 67) (Fig. 6B,C). Furthermore, injection of the recombination reporter into wild-type embryos (N = 57) and subsequent heat shock induction did not generate any β-galactosidase–positive cells, demonstrating the requirement of Cre-mediated recombination for the observed positive cells (data not shown). Hence, the EGFP–Cre fusion protein conditionally expressed in this transgenic line is capable of catalyzing site-specific recombination of two independent recombination reporter constructs.
Figure 6. Detection of β-galactosidase after heat shock induction of EGFP-Cre. A: Detection of expression from final lacZ recombination product (depicted at the top of the panel) in mosaic transgenic embryos at 24 hours postfertlization (hpf). B: Detection of β-galactosidase-positive (blue) cells after heat induction of a lacZ reporter injected HSP70-EGFP-cre transgenic embryo at approximately 42 hpf. C: Enlargement of boxed region of B.
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Whereas the previous two experiments demonstrated the ability of the EGFP-cre transgenic line to recombine injected plasmids, it is ultimately important to verify that the transgenic line is capable of recombining chromosomally integrated lox sites. Hence, the EGFP-cre transgenic line was crossed to a pCMV-loxH-STOP-loxP-EGFP transgenic reporter line (Fig. 1D). Unfortunately, expression of the reporter from the CMV promoter was extinguished in this generation of the transgenic line. PCR analysis, however, was capable of detecting the expected site-specific recombination product subsequent to heat shock-induction. As shown in Figure 7, for the EGFP-cre Tg/+ × pCMV-loxH-STOP-loxP-EGFP reporter cross, PCR amplification of the unrecombined transgene produces two products of 4.0 and 1.6 kb (Fig. 7A), while the recombined transgene yields only a 2.8-kb fragment with the same PCR primers (Fig. 7B). Importantly, the 2.8-kb recombinant PCR product is only detected after heat shock induction (Fig. 7C). The parental (unrecombined) fragments of 4.0 and 1.6 kb are detected at all time points due to the use of pools of embryos (20–30 embryos; half of which do not contain the HSP70-EGFP-cre transgene); hence, a subset of these embryos cannot recombine the transgene. Samples were taken at various time points subsequent to heat shock induction to determine how rapidly the production of EGFP-Cre could induce recombination. The recombined product was first apparent at 2 hr post–heat shock induction (Fig. 7C), and increasing amounts of recombined product were detected from 4, 8, and 12 hr post–heat shock (data not shown) through 24 hr post–heat shock (Fig. 7C). DNA sequencing of the recombined product at all three time points confirmed the accuracy of the Cre-mediated recombination between the loxH and loxP sites in the reporter. Furthermore, Cre-mediated recombination only occurred in pools of embryos from which PCR analysis could detect the presence of the HSP70-EGFP-cre transgene (Fig. 7C). Finally, PCR analysis and DNA sequencing determined that the recombined reporter was stably inherited in the subsequent generation (data not shown).
Figure 7. Detection of Cre-mediated site-specific deletion by polymerase chain reaction (PCR) analysis. A: Cartoon of two expected unrecombined PCR products. B: Cartoon of expected recombined PCR product. C: Analysis of recombination in EGFP-cre Tg/+ × pCMV-loxH-STOP-loxP-EGFP reporter Tg/+ cross and confirmation of presence of EGFP-cre Tg. “H2O” refers to the water (no DNA) control PCR reaction, and “no hs” refers to genomic DNA isolated from the transgenic cross in the absence of heat shock induction.
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Because smaller PCR products are preferentially amplified over large products using the same primers and pools of embryos were used for this experiment, the relative levels of recombined and unrecombined products cannot be accurately assessed in this experiment. Therefore, to determine the true efficiency of Cre-mediated recombination, we performed individual embryo PCR on offspring from a HSP70-EGFP-cre Tg/+ × pCMV-loxH-STOP-loxP-EGFP reporter cross. Embryos were heat shocked at 24 hpf and EGFP-Cre–positive and EGFP-Cre–negative embryos were harvested for PCR at 48 hpf. Of the embryos that contained both the EGFP-cre and the reporter transgenes, we found that 100% (N = 139) contained the recombined reporter product. In addition, a recombined reporter product was not observed in those EGFP-Cre–negative embryos that only contained the reporter transgene (N = 24). Therefore, the induction of the HSP70-EGFP-cre transgene is capable of relatively rapidly recombining chromosomally integrated lox sites after heat shock induction with incredibly high efficiency.
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- EXPERIMENTAL PROCEDURES
Cre is a highly efficient site-specific recombinase isolated from bacteriophage P1 (Hamilton and Abremski, 1984; Hoess et al., 1984). Cre mediates recombination between two 34-bp loxP sites (or variants thereof), such that recombination between directly repeated loxP sites leads to deletion of the intervening DNA and recombination between inverted loxP sites leads to inversion of the intervening DNA (Nagy, 2000). Cre recombinase can also be used to generate specific chromosomal translocations (Smith et al., 1995; Van Deursen et al., 1995), large scale chromosomal deletions (Ramirez-Solis et al., 1995; Li et al., 1996), or repeated high-frequency insertion of transgenes into the same genomic location (Fukushige and Sauer, 1992; Call et al., 2000). Hence, Cre has become a genomic engineering tool of vast utility in murine studies. Given the possible uses of these recombination events in zebrafish, we examined the feasibility of Cre expression and site-specific recombination.
A heat shock promoter-driven EGFP–cre fusion transcript was efficiently induced in a single-copy transgenic line and the resulting fusion protein catalyzed site-specific recombination of two separate, injected loxP and lox511 reporter constructs. Furthermore, the fusion protein promoted recombination of chromosomally integrated loxP and loxH sites in a transgenic line beginning 2–4 hr post–heat shock induction. Thus, the Cre recombinase is capable of recombining at least three different flavors of lox sites in zebrafish, suggesting that simultaneous deletion or inversion of several different transgenes carrying different variant of lox sites is possible.
Two independent experiments demonstrated that the Cre-mediated recombination was highly efficient. First, transient injection assays using the hcRFP reporter revealed that 62 to 70% of the EGFP-Cre–positive embryos catalyzed the recombination and expressed the reporter, which is comparable to 78% of the embryos injected with the recombined reporter exhibiting hcRFP expression (Table 3). Thus, the Cre-mediated production of mosaic hcRFP-expressing embryos was 90% of the level produced by injection of the previously recombined hcRFP reporter. A more appropriate test of the efficiency of EFGP-Cre was our PCR screen on 139 individual embryos that contained both the HSP70 EGFP-cre transgene and the pCMV-loxH-STOP-loxP-EGFP reporter transgene and the result that all 139 (100%) contained a recombined product. Taken together, these data indicate that the high efficiency of Cre-mediated recombination reported in other model systems can be achieved in zebrafish with the HSP70 EGFP-cre transgenic line.
Our transient reporter assays using the hcRFP reporter revealed that 3–4% of the injected EGFP-Cre embryos showed hcRFP-expressing cells, even in the absence of heat shock induction. This low level of recombination seen in the un-induced group may represent “leakiness” in the HSP70 promoter, possibly due to stressors other than heat or to promiscuous expression of HSP70 in certain tissues. In this vein, the original HSP70-EGFP transgenic line was reported to be “leaky” in the developing zebrafish eye (Halloran et al., 2000). Although we have not observed “leaky” EGFP-Cre expression in the eye or lens with the HSP70 EGFP-cre line, we have, in a few clutches, observed low-levels of EGFP in the developing notochord in un-induced embryos. Again, we attribute this either to this promoter being sensitive to other stressors besides heat in certain tissues or to promiscuous expression of HSP70 in the developing zebrafish notochord. This occasional EGFP expression in the notochord of uninduced embryos may account for the low level of hcRFP expression in the uninduced embryos, rather than a Cre-independent recombination event of the hcRFP reporter.
Rarely, Cre experiments have detected cytotoxicity in mammalian systems (Schmidt et al., 2000; Loonstra et al., 2001). Presumably, these results involve the utilization of the cryptic, yet functional, loxP sites that have been detected in mammalian genomes (Thyagarajan et al., 2000). Careful examination in tissue culture cells has revealed a dose-dependence to the cytotoxicity, with significant effects only occurring at very high levels of Cre expression (Loonstra et al., 2001). Experiments showing toxicity in mice involved expression of Cre from the protamine promoter. This highly active promoter produced Cre, while the histones were being replaced by protamines for compaction into the sperm head. Hence, the high level of Cre expression as well as the potentially sensitive form of chromosomes at this developmental stage may both contribute to the observed toxicity. In general, however, mouse lines expressing Cre have been relatively easy to generate and maintain as demonstrated by the current availability of lines on the Web site maintained by the Nagy laboratory (http://www.mshri.on.ca/nagy/Cre-pub.html).
Our studies suggest that induction of Cre expression does not have a significant adverse effect on the viability of transgenic zebrafish. Two generations of heat shock–induced offspring from the first HSP70-EGFP-cre founder have survived to sexual maturity, demonstrating that low level Cre expression is compatible with survival and absence of developmental defects. Moreover, 53% (90 of 167) of the progeny from a Tg/+ outcross to a wild-type fish carry the transgene, with both male and female offspring carrying the transgene being fertile subsequent to heat shock induction of EGFP-Cre. Finally, we are currently raising third-generation larvae and have not found any problems with inducing the expression of the transgene.
We demonstrated the successful generation of an inducible, stable Cre transgenic line in zebrafish. Although the ability to use Cre in numerous systems (Nagy, 2000; Werdien et al., 2001) suggested Cre use should be feasible in zebrafish, it remained important to experimentally demonstrate such feasibility and to check for cytotoxicity. Our initial studies suggest cytotoxicity will not be a critical issue for use of Cre in zebrafish. We are able to vary the amount of EGFP–Cre fusion protein generated by varying the length of heat shock and temperature used for the induction allowing optimization of the amount of protein for various recombination schemes. In conclusion, this study demonstrates that, in principle, the myriad of reverse genetic techniques for which Cre-mediated site-specific recombination is currently used in murine studies (Nagy, 2000; Branda and Dymecki, 2004) should be readily applicable in zebrafish as well. In addition, Cre-mediated recombination may serve either as an alternative means to or in conjunction with the GAL4-UAS system to spatially and temporally control transgene expression in zebrafish.