The transfer of full-length htt cDNA into a heterologous locus is a major challenge. Typically, when an insertion into a BAC is made by Red/ET recombination, the DNA fragment to be inserted is PCR-amplified with primers harboring 50 bp overhangs that mediate integration into the BAC at the intended position (20). In the present case, applying this strategy was impossible because of the size and fidelity limitations of PCR. The size of full-length htt cDNA is close to 10 kb and its polyQ region is very prone to variation in the number of CAG repeats. Furthermore, the CAG-repeat section in the htt cDNA is followed by a proline-rich region that extends across 120 bp and has a GC-content of over 80%. We used chimeric oligonucleotides comprising a primer binding site and a bifunctional homology arm in the PCR amplification of antibiotic resistance cassettes and thus introduced BAC-specific 50 bp homology arms on either side of the htt cDNA. Amplification of htt cDNA was thus submitted entirely to the bacterial replication machinery. While the concept of bifunctional homology arms has previously been presented in the literature as part of comprehensive recombineering studies (31-35), our study demonstrates for the first time how the concept can be applied to the mobilization of a complex cDNA and its transfer into a heterologous genomic locus.
Materials and methods
E. coli HS996 and DH10B were cultured aerobically using Luria-Bertani (LB) broth and agar. Media were supplemented with ampicillin, zeocin, chloramphenicol, or tetracycline as appropriate, to final concentrations of 100, 50, 15, or 3 µg/mL. Kanamycin was added to culture media at final concentrations of 50 µg/mL for high-copy plasmids or 15 µg/mL for BACs.
Electroporations were performed in an Eppendorf Electroporator 2510 (Eppendorf, Hamburg, Germany) in chilled electroporation cuvettes (1 mm gap) at 1350 V, 10 µF, and 600 Ohms. Following electroporation, cells were taken up in 1 mL LB medium and were allowed to recover for 1 h at the appropriate temperature before plating on selective agar. Bacterial cells were typically electroporated with 200 ng of linear DNA. Electrocompetent cells for retransformation were prepared as described elsewhere (36).
BAC DNA purification
For analytical BAC preparations, overnight cultures of E. coli DH10B harboring the BAC of interest were sedimented and treated with buffers P1, P2, and P3 of the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The obtained cell lysate was cleared by centrifugation and transferred into a reaction tube containing 0.7 volumes of isopropanol. Precipitated BAC DNA was sedimented by centrifugation, washed once with 70% (w/v) ethanol, suspended in ddH2O, and stored at -20°C until use.
High-quality BAC DNA for pulsed-field gel electrophoresis was prepared using the QIAGEN Plasmid Maxi Kit (Qiagen), according to the manufacturer's instructions. DNA concentrations were determined on a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA), assuming that an A260 of 1 is equivalent to 50 µg/mL dsDNA.
Red/ET recombination was performed according to standard protocols of Gene Bridges GmbH (Heidelberg, Germany). Briefly, bacterial cells harboring pRed/ET were cultured aerobically in 1.4 mL LB medium at a temperature of 30°C. At an OD600 of approximately 0.3, expression of red genes was induced by the addition of 50 µL 10% (w/v) arabinose. At the same time, the temperature was increased to 37°C to ensure maximal expression and activity of the recombination proteins and prohibit replication of pRed/ET. After 45 min, cells were sedimented, washed twice with ice-cold 10% (v/v) glycerol, and electroporated with substrate DNA. Samples from which L-arabinose was omitted were used as negative controls.
Pulsed-field gel electrophoresis (PFGE)
Prior to electrophoresis, 1 µg of BAC DNA was digested in a total volume of 20 µL 1× restriction buffer containing 10 U of restriction endonuclease XhoI for 1 h at 37°C.
Pulsed-field gel electrophoresis was performed in the two state mode in 0.5× TBE buffer with an included angle of 120°, a gradient of 6 V/cm and a runtime of 16 h. The initial and final switch times were 2.16 s and 3.49 s, respectively, with linear ramping. Restriction fragments were resolved on a 1% (w/v) agarose gel, which was stained with 2 µg/mL ethidium bromide in ddH20 for 20 min, then destained in ddH20 for 40 min before documentation. UltraRanger 1kb DNA Ladder (Norgen Biotek Corp., Thorold, ON, Canada) and Low Range PFG Marker (New England Biolabs, Ipswich, MA, USA) were used as molecular weight markers.
Transgenesis and genotyping
The vectors were prepared for injection as described by Gama-Sosa et al. (37) and injected into fertilized eggs of C57BL/6Jmice at the transgenic facility of Mount Sinai School of Medicine. Transgenic animals were identified by performing PCR on genomic DNA purified from tail biopsies using the REDExtract-N-Amp Tissue PCR kit (Sigma, St. Louis, MO, USA). The primers amplified downstream of the CAG repeat, across the exon 2–3 junction: sense 5′-CCgCTgCACCgACCAAAgAA–3′ and antisense 5′-gCATTCgTCAg-CCACCATCC–3′. Additionally, tail biopsies were sent to Laragen (Los Angeles, CA, USA) for genotyping by PCR as well as determination of the CAG repeat length. Mice were maintained on a C57BL/6J background.
Protein Expression Analysis
Protein levels were measured by Western blot analysis of protein extracted from regionally dissected brain tissue. Soluble protein was extracted from tissue in 20 mM Hepes (pH 7.6), 150 mM NaCl, 0.5 mM EDTA, and 0.5% Triton X-100, supplemented with 1× Complete proteaseinhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany), 1 mM PMSF, 50 mM NaF, and 1 mM Na-orthovanadate. Protein levels in the extracts were determined using the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). peqGOLD Protein-Marker VII (PEQLAB, Wilmington, DE, USA) was used as a molecular weight marker. Transgenic Htt was detected with 1C2 antibody (1:1000, MAB1574; Chemicon, Temecula, CA, USA). Blots were developed using Amersham ECL Western blot detection reagents (Piscataway, NJ, USA).
Enzymes, kits and reagents
The PCR Extender System (5PRIME GmbH, Hamburg, Germany) was used for PCR amplifications, which were performed in a total volume of 50 µL 1× Tuning Buffer with Mg2+ containing 100 ng template DNA, 200 µM each dNTP, 200 nM each primer, and 2.5 U PCR Extender Polymerase Mix. Temperature cycling comprised an initial denaturation for 2 min at 94°C, followed by 30 cycles of 94°C for 20 s, 54°C for 20 s, 72°C for 1 min/kb, and a final elongation step for 5 min at 72°C. Primers were obtained from BioSpring (Frankfurt, Germany) and sequencing was performed by GATC Biotech (Konstanz, Germany). Restriction enzymes were purchased from Fermentas (St. Leon-Rot, Germany). Antibiotic resistance cassette A002, expression plasmid pRed/ET and the Counter-Selection BAC Modification Kit were obtained from Gene Bridges. HyperLadder I (Bioline, Taunton, MA, USA) was used as a molecular weight marker for standard agarose gel electrophoresis and chemicals and antibiotics were obtained from Applichem (Darmstadt, Germany). Gel extractions and plasmid purifications were performed using the MinElute Gel Extraction Kit and QIAprep Spin Miniprep Kit (Qiagen), according to the manufacturer's instructions.
Results and discussion
This study evaluates a recombineering protocol based on chimeric oligonucleo-tides for the generation of htt BDNF-BAC transgenes. (A graphic representation of BDNF-BAC RP23–106l19 and partial sequences of the bdnf gene locus and htt cDNA can be found in Supplementary Figure 1.) Figure 1 illustrates the workflow for the generation of the BDNF-BAC transgenes. Central to the protocol are chimeric oligonucleotides comprising a primer binding site and a bifunctional homology arm, bearing on its distal end, 50 nucleotides mediating integration into the substrate plasmid and on its proximal end, 50 nucleotides mediating integration into the BAC, wherein a unique SacII restriction site positioned next to the BAC-specific homology arms allows their exposure by restriction digest. The oligonuc-leotides were used as primers in the amplification of antibiotic resistance cassettes, which were inserted into plasmid pFL98Q (Supplementary Table 1) on either side of htt cDNA. The use of an antibiotic resistance marker in every Red/ET recombination step is required because of the efficiency of Red/ET recombination, which lies in the range of 10−3 to 10−4 (38).