Constructs and Injections
We have cloned the Tol2 transposon from medaka DNA by using PCR primers designed based on Tol2 sequence (GenBank accession no. D84375). Cloning of Tol2 was performed in two steps using PCR primers: CATGCGGGCCCAGAGGTGTAAAGTACTTGAGTA and GCGCAAGCGGCCGCTTGAGACTAGGTTAAGTA for the 5′ end of the element and CGCGCTCGAGCAGAGGTGTAAAAAGTACTCAA and ATATAGGCGGCCGCCTGTGTTTCAGACACCA for the 3′ end. The first PCR product was cloned into pBK-CMV (Stratagene, USA) using ApaI and NotI restriction sites incorporated into the primers, followed by cloning the 3′-end product into the construct using NotI and XhoI restriction sites so that both PCR fragments were joined at the internal NotI site. The construct containing EGFP (Clontech Laboratories, USA) under 2.2-kb promoter of keratin 8 (krt8) gene (GenBank accession no. AF440690) was obtained from Dr. Zhiyuan Gong from the National University of Singapore. A fragment containing the EGFP gene and only 460 bp of the krt8 promoter was amplified using this plasmid as a template and primers ACAATGCAACTGTTCAGCTCA and ATGGCTGATTATGATCTAGAG. The product was digested by NotIand cloned into the Tol2 construct between EcoRV and NotI sites. The final enhancer trap construct (Fig. 1) consists of 2,671 bp of Tol2 5′ end, 529 bp Tol2 3′ end, and the mini-krt8 promoter-EGFP fragment (1,200 bp long). Transposase cDNA was cloned from medaka cDNA using the primer: ACGTGAGCTCACATCTATTACCACAATGCAC. We used mMESSAGE mMACHINE T7 kit (Ambion) for synthesis of in vitro transcribed capped transposase RNA and RNeasy Mini Kit (QIAGEN, Germany) for RNA cleanup. A total of 5–10 pg of plasmid DNA with 25–50 pg of in vitro synthesized transposase mRNA were coinjected into zebrafish embryos at the one- to two-cell stage. The actual concentration of RNA was empirically adjusted to produce 50% embryo survival rate.
TAIL-PCR was performed according to Liu and Whittier (1995) using the following set of primers: Toil5′-1, GGGAAAATAGAATGAAGTGATCTCC; Toil5′-2, GACTGTAAATAAAATTGTAAGGAG; Toil5′-3, CCCCAAAAATAATACTTAAGTACAG; Toil3′-1, CTCAAGTACAATTTTAATGGAGTAC; Toil3′-2, ACTCAAGTAAGATTCTAGCCAGA; Toil3′-3, CCTAAGTACTTGTACTTTCACTTG; AD-3, WGTGNAGNANCANAGA; AD-5, WCAGNTGWTNGTNCTG; AD-6, STTGNTASTNCTNTGC; AD-11, NCASGAWAGNCSWCAA.
The following primer mixtures (containing 1.5 μM specific primer and 10 μM AD primer) were prepared: for primary PCR: Toil5′-1/AD-3, Toil5′-1/AD-5, Toil5′-1/AD-6, Toil5′-1/AD-11, Toil3′-1/AD-3, Toil3′-1/AD-5, Toil3′-1/AD-6, Toil3′-1/AD-11; for secondary PCR: Toil5′-2/ AD-3, Toil5′-2/AD-5, Toil5′-2/AD-6, Toil5′-2/AD-11, Toil3′-2/ AD-3, Toil3′-2/AD-5, Toil3′-2/AD-6, Toil3′-2/AD-11; for tertiary PCR: Toil5′-3/ AD-3, Toil5′-3/AD-5, Toil5′-3/AD-6, Toil5′-3/AD-11, Toil3′-3/AD-3, Toil3′-3/AD-5, Toil3′-3/AD-6, Toil3′-3/AD-11. A total of 4 μl of primer mixtures were added to PCR reaction (total volume 20 μl).
Cycle settings were as follows. Primary: (1) 94°C, 2 min; (2) 94°C, 30 sec; (3) 62°C, 1 min; (4) 72°C, 2.5 min; (5) go to “cycle 2” 4 times; (6) 94°C, 30 sec; (7) 25°C, 3 min; (8) ramping 0.3°/sec to 72°C; (9) 72°C, 2.5 min; (10) 94°C, 10 sec; (11) 61°C, 1 min; (12) 72°C, 2.5 min; (13) 94°C, 10 sec; (14) 61°C, 1 min; (15) 72°C, 2.5 min; (16) 94°C, 10 sec; (17) 44°C, 1 min; (18) 72°C, 2.5 min; (19) go to “cycle 10” 14 times; (20) 72°C, 5 min.
A total of 2 μl of the primary reaction was diluted in 25 μl of water and 2 μl of the mixture was added to the secondary reaction. Secondary: (1) 94°C, 10 sec; (2) 61°C, 1 min; (3) 72°C, 2.5 min; (4) 94°C, 10 sec; (5) 61°C, 1 min; (6) 72°C, 2.5 min; (7) 94°C, 10 sec; (8) 44°C, 1 min; (9) ramping 1.5°/sec to 72°C; (10) 72°C, 2.5 min; (11) go to “cycle 1” 14 times; (12) 72°C, 5 min.
A total of 2 μl of the secondary reaction was diluted in 25 μl of water, and 2 μl of the mixture was added to the tertiary reaction. Tertiary: (1) 94°C, 15 sec; (2) 44°C, 1 min; (3) ramping 1.5°/sec to 72°C; (4) 72°C, 2.5 min; (5) go to “cycle 1” 29 times; (6) 72°C, 5 min.
Products of the secondary and tertiary reactions were separated by using 1.8% agarose gel. The individual bands from the “band shift” pairs were cut from the gel and purified by using QIAquick Gel Extraction Kit (QIAGEN, Germany), and sequenced by using ABI Cycle Sequencing chemistry (PE Applied Biosystems, CA) and an ABI Prism 310 Genetic Analyzer with Data Collection Software (PE Applied Biosystems, Foster City, CA) supplied by the producer.
RNA In Situ Hybridization
Whole-mount in situ hybridization using antisense RNA probes labeled with digoxigenin (Roche, USA) was carried out as previously described by Oxtoby and Jowett (1993). Zic6 and fd54c09 cDNA templates, used to generate RNA probes, were amplified using OneStep RT-PCR kit (Qiagen, Germany) with the following pairs of primers correspondingly: zic5′-GGGACAAATCTGTCAGCAGCA and T7zic3′-ATTGTAATACGACTCACTATAGGTATGCCACAAACCTATCAACT; psc5′-CACATACAGACATCATCTTTGCAC and T7psc3′-ATATAATACGACTCACTATAGGGATGCAGCCAGTGGCTGTAA. After in situ hybridization, stained embryos were mounted in phosphate buffered saline–glycerol and viewed under AxioPlan 2 microscope (Zeiss, Germany). Images were taken with AxioCam digital camera (Zeiss, Germany) and processed using Adobe Photoshop 5.5 software.
Southern Blot Hybridization
After digestion by BamHI or EcoRI, DNA was transferred to positively charged nylon membrane (Roche Applied Science) by capillary blotting (Sambrook et al., 1989) and cross-linked by ultraviolet irradiation.
The DNA probe for EGFP was labeled with digoxigenin (Roche Applied Science) using PCR digoxigenin (DIG) synthesis kit. We used DIG EasyHyb DIG Wash and Block Buffer Set for hybridization, alkaline phosphatase labeled anti-DIG antibody, and CDP-Star chemiluminescent substrate (Roche Applied Science, USA) for detection of the hybridized probe. Hybridization and detection was carried out as described in the manufacturer's User's Guide.
Analysis of Tol2 Excision Sites
Heterozygous ET33 embryos were injected with 25–50 pg of transposase mRNA and raised to maturity. The adult fish were out-crossed to the wt, and their progeny (F1) were analyzed for new GFP patterns. Individual embryos carrying retransposed element (1 from each of the 12 founders) were used for PCR with primers on either site of the original insertion: GAGTCAGCTCAGTACTCATG and TACTTCATCTGCCGGGTCTT.
The PCR product (464 bp in the wt) was sequenced in both directions. Because founders were heterozygous for the donor, the individual progeny containing retransposed element could be either heterozygous for the “recovered” donor site or may not contain the donor site due to segregation. Fish containing a footprint produced a mixed sequencing electropherogram starting from the footprint. Because there is no difference between wt sequence and the product of “precise precision,” obtaining the wt sequence in such test could not be directly used to confirm “precise excision” (it could be due to segregation).