Introduction

The recent completion of human and mouse whole genome sequencing projects has revolutionized the way we try to understand biology and will certainly have significant influence on our insights into human diseases at a systemic level. Although the availability of the genomic information provides a great framework for biologists to address a broad range of scientific questions, most of mammalian genes have not been extensively studied yet. In fact, 30%–50% of genes fall into the uncharacterized category (1). Forward genetics represents a classical approach to uncover gene function. One powerful forward genetics strategy is insertional mutagenesis using DNA mutagens, such as retroviruses and transposable elements, which integrate into host genome DNA and can up-regulate or disrupt gene function. Through the identification of the insertion sites of these mutagens, a gene can be assigned a certain phenotype-related function. Recently, insertional mutagenesis has been demonstrated to be a fruitful strategy in genome-wide analysis of cancer genes (2-8). The BXH-2 strain of mice develops acute myeloid leukemia (AML) at a high incidence, providing a good model to identify leukemia genes. The BXH-2 AML arises from infection by a murine leukemia virus (MuLV), which cannot only act as an insertional DNA mutagen to cause leukemia, but also serves as a tag for leukemia-associated genes (9).

Mapping proviral insertion sites (PISs) is important not only for an appreciation of the molecular mechanism of integration, but also for characterization of cellular gene functions using insertion mutagenesis strategy. Several methods have been developed for this purpose, including genomic DNA library screening (10), ligation-mediated PCR (LM-PCR) (3), inverse PCR (11), viral insertion site amplification (VISA) technique (12), and single nucleotide polymorphism (SNP)-based mapping (13,6). Although these methods have been useful and generated large amounts of PISs, their inherent limitations posed either by uneven restriction site distribution, low cloning efficiency, excessive laborious work, or nonspecific amplification need to be overcome in order to facilitate functional genomic studies. Here, we described a PCR-based method allowing for less laborious, less biased, and more efficient mapping of PISs. We applied the method to a large-scale identification of PISs associated with BXH-2 AML. We also assessed the PIS pattern in leukemia cells undergoing drug selection.

Materials and Methods

Cell Lines and Culture

The BXH-2 AML cell lines used in this study were generated and maintained routinely as previously reported (14,15). Highly Ara-C-resistant cell line B117H was derived by repetitive exposure of the parental cell line B117 to increasing doses of Ara-C (Pfizer, Kalamazoo, MI, USA) (16). The passage control cell line B117P was simply grown and passaged in regular ASM medium in parallel with selection of drug-resistant cells. The Ara-C sensitivity of control cells and resistant cells was verified by a cytotoxicity assay (16).

Southern Blot Analysis

Genomic DNA was prepared and analyzed by Southern blot analysis as described before (16). Briefly, 10 µg genomic DNA were digested with the restriction enzymes PvuII and resolved on a 0.9% agarose gel. The gel was then subjected to depurination, denaturation, neutralization, and blotting. The 400-bp B-ecotropic MuLV-specific probe, pEnv, was generated by cutting the plasmid pAKV5 with restriction enzyme XmaI (17) and labeled with α-³²P-dCTP using the random priming labeling approach. The blots were blocked with prehybridization solution for 4 h at 65°C and hybridized with the labeled probes in hybridization solution at 65°C overnight. After nonspecific binding was removed with the third washing solution [0.25× single-strand conformation polymorphism (SSCP), 0.1% sodium dodecyl sulfate (SDS)], the blots were exposed to X-ray films (Eastman Kodak, Rochester, NY, USA) at −70°C with intensifying screens.

SplinkTA-PCR Protocol

For SplinkTA-PCR (STA-PCR), genomic DNA was digested with 7.5 U TaqI per microgram DNA at 65°C for 4 h followed by another 4-h digestion with 7.5 U BspLU11I and 10 U BclI (Roche Diagnostics, Indianapolis, IN, USA) per microgram DNA at 48°C. In some experiments, different restriction enzyme cocktails were used (see Results and Discussion section). The digestion products were purified with QIAquick® PCR Purification kit (Qiagen, Valencia, CA, USA) according to the manufacturer. Briefly, 250 µL Buffer PB (Qiagen) were added to each sample, and the mixture was applied to the minicolumns, centrifuged at 11,000× g for 30 s, washed with 750 µL Buffer PE (Qiagen) at 11,000× g for 30 s, and centrifuged for an additional 60 s at maximum speed. The DNA was eluted with H₂O. For adenosine addition reactions, each purified, digested DNA sample was incubated with 1× final concentration of PCR buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, and 2.5 U Taq DNA polymerase (BIOLASE Technology, Irvine, CA, USA) at 72°C for 20 min, followed by another purification using the QIAquick kit. SplinkTA was made by mixing equal molar amounts of oligonucleotides splinkerette (5′-CATGGTTGTTAGGACTGGAGGGGAAATCAATCCCCT-3′; the hairpin oligonucleotide) and PrimerLongTA (5′-CCTCCACTACGACTCACTGAAGGGCAAGCAGTCCTAACAACCATGT-3′; the oligonucleotide with an extra T) and incubating for 5 min at 80°C before allowing it to slowly cool down to room temperature (20°−25°C). Then an appropriate amount of SplinkTA was ligated to the extension products using T4 DNA ligase (Promega, Madison, WI, USA) by incubation at 16°C for 16–20 h. Using 1 µL ligation products as templates, primary PCR was done by adding 1× final concentration of PCR buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 20 pmol primer AKVp7711 (specific to BXH-2 proviral Env gene), 20 pmol primer P-short (complementary to PrimerLongTA), and 2.5 U Taq DNA polymerase (BIOLASE Technology). PCRs were performed at 95°C for 1 min 30 s, followed by 10 cycles of 95°C for 5 s; 70°C for 3 min 10 s with decreasing 0.5°C each cycle; 20 more cycles of 95°C for 5 s; 65°C for 3 min 10 s; and final extension at 70°C for 10 min. In secondary PCR, all components in each reaction are the same as those in primary PCR, except for using primary PCR products as templates and replacing the pair of primers with nested primers AKVp8712 [specific to BXH-2 proviral long terminal repeat (LTR)] and Pnest (complementary to PrimerLongTA). The amplification parameters were changed to denature at 95°C for 1 min, followed by 11 cycles of 94°C for 15 s; 70°C for 2 min 40 s with decreasing 0.6°C each cycle; 25 more cycles of 94°C for 15 s; 64°C for 30 s; 70°C for 2 min 10 s; and final extension at 70°C for 10 min. After electrophoresis on a 1.5% agarose gel, individual secondary PCR bands were taken and subsequently cloned into TOPO-TA® vector with TOPOTA Cloning kit (Invitrogen, Carlsbad, CA, USA) for sequencing. Additional primer sequences are available upon request.