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Figure 2. Transgene detection in serial dilutions. Sperm containing one copy each of transgene was serially diluted into wild-type sperm solution. Modified
HotSHOT was used to isolate DNA from each dilution, and then PCR was performed for detection of transgene. Lane A, 1-kb plus DNA ladder. DNA source
for lanes B–I as follows: B, undiluted transgene-containing sperm; C–G, successive 4-fold sperm dilutions (1×, 4×, 16×, 64×, 256×, and 1024×); H, wild-type
DNA; I, water. PCR fragment size, 311 bp.
dATP, dCTP, dGTP, and dTTP, and 0.1 founders that are mosaic for the as described above. The transgene
U Taq DNA polymerase. Reactions transgene, most notably in germ cells. was detectable even when diluted
were amplified in a Model PTC-225 The most efficient method for identi- greater than 1000-fold (Figure 2),
Peltier Thermal Cycler (MJ Research, fying founders is by PCR of genomic indicating that the modified HotSHOT
San Francisco, CA, USA) using the DNA isolated from sperm. Thus, method can be used to detect trans-
following conditions: 95°C for 3 min; DNA isolation methods must provide genic males carrying the transgene in
35 cycles of 95°C for 30 s, 60°C for DNA of sufficient quality to allow as few as 0.1% sperm.
30 s, and 72°C for 1 min; followed by for detection of the transgene when In conclusion, this method is rapid,
72°C for 10 min. Fifteen microliters diluted in wild-type DNA. To demon- reliable, and cost-efficient for the
each reaction were loaded onto a 1% strate that the modified HotSHOT isolation of PCR-quality DNA from
SeaKem LE agarose gel (Cambrex, technique meets this requirement, zebrafish tissues. The DNA solutions
East Rutherford, NJ, USA) containing we serially diluted sperm from males are stable at 4°C for at least 3 months
ethidium bromide and electrophoresed homozygous for the lck::EGFP and can be kept frozen at -20°C for
at 6 V/cm for 45 min in 1× Tris-borate- transgene into wild-type sperm and longer storage. We anticipate that
EDTA (TBE) buffer Tris 90 mM, then isolated DNA from the dilutions. adoption of this protocol among the
Boric Acid 90 mM, EDTA 2 mM, Five microliters sperm from trans- zebrafish community will speed
pH 8.2 (Sigma-Aldritch, St. Louis, genic males and 10 μL sperm from genotyping, increasing the efficiency
MO). The gel was imaged using a wild-type Tübingen fish were added of the identification of transgenic
Bio-Rad Gel Doc 2000 apparatus and to 50 μL and 100 μL 50 mM NaOH, animals, the mapping of mutants, and
the Quantity One software (both from respectively. The solutions were other PCR-based applications.
Bio-Rad Laboratories, Hercules, CA, briefly vortex mixed. The wild-type
USA) (Figure 1). A band matching sperm solution was separated into
the target size of 311 bp was present 15-μL aliquots. Five microliters ACKNOWLEDGMENTS
in each lane, indicating that the DNA transgenic sperm solution were added
isolated from each zebrafish tissue was to the first aliquot. Serial dilutions This research was supported
of adequate quality for reliable PCR. were then accomplished using 5 μL in part by the Children’s Health
Transgenic zebrafish are generated each dilution into the next aliquot, Research Center, University of Utah
by injecting DNA constructs into resulting in a 1:1024 final dilution. (NDM, as a Primary Children’s
one-cell stage embryos that are DNA was isolated in each tube as Medical Center Foundation Scholar),
then raised to adulthood. Random described above. Five microliters each and also by a grant from the DANA
integration results in transgenic solution were used in 25-μL PCRs Foundation (NST).
612 ı BioTechniques ı www.biotechniques.com Vol. 43 ı No. 5 ı 2007
Benchmarks
COMPETING INTERESTS extraction from animal tissues for polymerase Received 14 August 2007; accepted
chain reaction. Electrophoresis 26:3081-3083. 18 September 2007.
STATEMENT
2. Truett, G.E., R.L. Mynatt, A.A. Truett,
J.A. Walker, and M.L. Warman. 2000.
The authors declare no competing Preparation of PCR-quality mouse genomic Address correspondence to Nikolaus
interests. DNA with hot sodium hydroxide and Tris S. Trede, Huntsman Cancer Institute,
(HotSHOT). BioTechniques 29:52-54.
2000 Circle of Hope, Salt Lake City, UT,
3. Langenau, D.M., A.A. Ferrando, D. Traver,
J.L. Kutok, J.P. Hezel, J.P. Kanki, L.I. Zon, 84112 USA. e-mail: nikolaus.trede@hci.
REFERENCES A.T. Look, and N.S. Trede. 2004. In vivo utah.edu
tracking of T cell development, ablation, and
1. Yue, G.H. and L. Orban. 2005. A simple and engraftment in transgenic zebrafish. Proc. Natl. To purchase reprints of this article, contact:
affordable method for high-throughput DNA Acad. Sci. USA 101:7369-7374. Reprints@BioTechniques.com