Splinkerette PCR for mapping transposable elements in Drosophila.
Transposable elements (such as the P-element and piggyBac) have been used to introduce thousands of transgenic constructs into the Drosophila genome. These transgenic constructs serve many roles, from assaying gene/cell function, to controlling chromosome arm rearrangement. Knowing the precise genomic insertion site for the transposable element is often desired. This enables identification of genomic enhancer regions trapped by an enhancer trap, identification of the gene mutated by a transposon insertion, or simplifying recombination experiments. The most commonly used transgene mapping method is inverse PCR (iPCR). Although usually effective, limitations with iPCR hinder its ability to isolate flanking genomic DNA in complex genomic loci, such as those that contain natural transposons. Here we report the adaptation of the splinkerette PCR (spPCR) method for the isolation of flanking genomic DNA of any P-element or piggyBac. We report a simple and detailed protocol for spPCR. We use spPCR to 1) map a GAL4 enhancer trap located inside a natural transposon, pinpointing a master regulatory region for olfactory neuron expression in the brain; and 2) map all commonly used centromeric FRT insertion sites. The ease, efficiency, and efficacy of spPCR could make it a favored choice for the mapping of transposable element in Drosophila.
Potter
Christopher J
CJ
Solomon H. Snyder Department of Neuroscience, Center for Sensory Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America. cpotter@jhmi.edu
Luo
Liqun
L
D000818:Animals; D004251:DNA Transposable Elements / Q000235:genetics*; D004330:Drosophila / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D016254:Mutagenesis, Insertional / Q000379:methods; D018034:Olfactory Receptor Neurons; D016133:Polymerase Chain Reaction / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics; D019076:Transgenes
D004251:DNA Transposable Elements; D014157:Transcription Factors
Genetics. 2004 Apr;166(4):1775-82
15126397
Nat Neurosci. 2009 Jan;12(1):53-9
19043409
Nucleic Acids Res. 1994 Aug 25;22(16):3429-30
8078781
EMBO J. 1989 Jan;8(1):211-7
2540958
Nat Protoc. 2006;1(4):2110-5
17487202
Development. 1993 Apr;117(4):1223-37
8404527
Curr Biol. 2004 Mar 23;14(6):449-57
15043809
Cell. 2007 Mar 23;128(6):1187-203
17382886
Genetics. 1996 Mar;142(3):907-14
8849896
Nat Genet. 2007 Aug;39(8):933-4
17660805
Proc Natl Acad Sci U S A. 1987 Dec;84(24):9123-7
2827169
Genetics. 1996 Dec;144(4):1673-9
8978054
J Cell Sci. 2004 Apr 15;117(Pt 10):1911-22
15090595
Science. 1988 Mar 4;239(4844):1121-8
2830671
Nat Protoc. 2007;2(11):2910-7
18007627
EMBO J. 1987 Dec 20;6(13):4125-35
2832152
Nucleic Acids Res. 1995 May 11;23(9):1644-5
7784225
Genetics. 1993 Mar;133(3):605-22
8384145
Science. 1982 Oct 22;218(4570):348-53
6289436
Genetics. 2007 Mar;175(3):1089-104
17179094
Genes Dev. 1989 Sep;3(9):1273-87
2558049
Curr Biol. 2008 Nov 25;18(22):1754-9
19013069
Science. 1982 Oct 22;218(4570):341-7
6289435
Insect Mol Biol. 1999 Nov;8(4):449-57
10634970
Nat Genet. 2008 Apr;40(4):476-83
18311141
Proc Natl Acad Sci U S A. 2008 Jul 15;105(28):9715-20
18621688
Nat Genet. 2004 Mar;36(3):283-7
14981521
Genome Biol. 2002;3(12):RESEARCH0084
12537573
Genetics. 1993 Jul;134(3):859-68
8394264
Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9485-9
7568159
Genet Res. 1994 Dec;64(3):167-81
7698641
J Comp Neurol. 1999 Mar 22;405(4):543-52
10098944
Nat Protoc. 2009;4(5):789-98
19528954
Genetics. 1988 Nov;120(3):621-3
2852134
Development. 1993 Jun;118(2):401-15
8223268
Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3312-7
17360644
J Neurobiol. 1997 May;32(5):443-56
9110257
Dev Cell. 2008 Feb;14(2):227-38
18267091