Huntington's disease (HD) is a fatal neurodegenerative disorder that is caused by a CAG repeat expansion encoding a polyglutamine tract in the huntingtin (htt) gene. None of the existing HD mouse models recapitulate the exact disease symptoms and course as it is seen in humans and the generation of further HD disease models is challenging because of the size and complexity of the htt gene locus. Starting from a single substrate plasmid harboring human htt cDNA comprising 98 glutamine (Q) residues, we applied Red/ET recombination to generate four BDNF-BAC transgenes harboring full-length or truncated (N171) htt cDNA comprising 98 or 15 Q residues. BDNF (brain-derived neurotrophic factor) is expressed in the cortical neurons projecting to the striatal medium spiny neurons, and was used to direct htt transgene expression to investigate the contribution of these cell types to HD.

Our method uses chimeric oligonucleo-tides comprising a primer binding site and a bifunctional homology arm. These oligonucleotides were used in the PCR-amplification of antibiotic resistance cassettes, which were inserted into the substrate plasmid adjacent to the htt cDNA. Using unique restriction sites, the cDNA was excised from the source plasmid and transferred into the BAC. The presented recombineering protocol will facilitate the generation of mouse models of HD and will be applicable to many other complex cloning exercises.

Huntington's disease (HD) is an autosomal dominant, uniformly fatal neurodegenerative disorder with a prevalence of approximately 4–10 in 100,000 (1). The disease manifests itself with a variety of symptoms, including motor and cognitive deficiencies as well as psychiatric disturbances.

The gene responsible for the disease is huntingtin (htt), which comprises 67 exons and occupies a stretch of DNA approximately 170 kb in size (2, 3). This corresponds to a cDNA length of approxi-mately 10 kb. In healthy individuals, the Htt protein typically harbors a stretch of between 6 and 36 CAG (Q) repeats near its N terminus. Expansion of this polyQ stretch causes HD, with the number of Q residues correlating with the age of onset and severity of the symptoms. Commonly, individuals that experience onset of the disease as adults harbor repeat expansions comprising 40 to 55 units, while repeat expansions comprising 70 units or above are invariably associated with the juvenile form of HD (4-6).

On the cellular level, the disease manifests itself in the degeneration of multiple neuronal subtypes, but striatal GABAergic projection neurons and cortical pyramidal neurons are uniquely vulnerable. Pathologically, the disease is characterized by increased nuclear locali-zation of mutant Htt and the formation of intranuclear inclusions and cytoplasmic aggregates comprised of small N-terminal fragments of mutant Htt (7).

A number of mouse models of HD have been generated with the goal of developing a useful model for therapeutic testing. Transgenic mouse models of HD comprise models harboring an N-terminal fragment of mutant htt or full-length mutant htt under the control of its endogenous regulatory machinery or a heterologous promoter. Among the fragment models, the R6/2 model is the most widely used. The inserted fragment comprises exon 1 of human htt, originally modified to harbor 145 CAG repeats (8, 9). A second fragment model, prp N-171, expresses the N-terminal 171 amino acids of mutant htt with an 82Q chain (10). Transgenic models harboring full-length mutant htt include the BACHD and YAC128 models (11, 12). Generally, in transgenic models, an increase in the severity of the symptoms is observed as the length of the CAG repeat and the expression levelof polyQ-htt increase, and as the length of the htt transgene decreases (13).

Knock-in mouse models in which the endogenous murine htt gene has been modified to harbor the pathogenic CAG expansion mimic the genetic defect seen in humans in the most accurate way possible, but display a very late-onset disease phenotype (14, 15). It is obvious that there is currently no single HD mouse model that replicates the exact disease course as it is seen in humans. As there are no effective treatments for HD, there is a strong demand for the development of further clinically relevant mouse models that can be used for therapeutic testing and the study of HD pathophysiology.

Red/ET recombination (recombineering) has become the method of choice when it comes to engineering large replicons, such as BACs or the E scherichia coli chromosome. The technology relies on in vivo homologous recombination mediated by the Rac-phage recE and recT genes (16, 17) or the phage lambda redα and redβ genes (16, 18-27). Typically, Red/ET recombination occurs between a linear and a circular reaction partner (17) and requires homology arms of approximately 50 bp in length (20), which are conveniently attached to the linear reaction partner by PCR.

We demonstrate the generation of four BDNF-BAC transgenes harboring full-length or truncated (N171) human htt cDNA comprising 98 or 15 CAG-repeats to be used for the generation of novel transgenic mouse models of HD in which cortical contributions to phenotype could be isolated. Htt cDNA was inserted into exon 2 of the bdnf genomic locus and was thus placed under the transcriptional control of the bdnf promoter. BDNF is a neurotrophic factor that is widely expressed in the developing and adult mammalian brain, including in the corticostriatal projection neurons. It contributes to the maturation, maintenance, or survival of many neuronal cell types, including the GABA-ergic medium-sized spiny striatal neurons (MSNs) that die in HD (28-30). Thus, expression of mutant Htt in these neurons would potentially constitute a model to isolate the contribution of pyramidal corticostriatal neurons to HD.

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

Bacterial culture

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.

Bacterial transformation

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

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).

Figure 1. Schematic diagram of the cloning strategy for generating the BDNF-BAC transgenes. (Click to enlarge)

Red/ET recombination was performed as follows: a zeocin resistance cassette and an FRT-flanked kanamycin/neomycin resistance cassette were PCR-amplifiedwith primers PCR1–5′ and PCR1–3′ or PCR2–5′ and PCR2–3′ (Supplementary Table 1). With a goal of generating a BAC transgene harboring truncated N171 98Q htt cDNA, the FRT-flanked neomycin resistance cassette was also amplified with primers PCR3–5′ and PCR3–3′, which contained homology arms mediating integration into full-length 98Q htt cDNA downstream of codon 246, thereby deleting the remaining htt cDNA. In a first Red/ET recombination step, 200 ng of PCR-product 1 were electroporated into E. coli cells harboring pRed/ET and pFL98Q to generate pFL98Q1. ≥200 ampicillin- and zeocin-resistant colonies were obtained from the electroporated cultures that had been induced with L-arabinose, while no colonies were obtained from the non-induced controls. Individual clones were propagated and plasmid DNA was isolated and analyzed by restriction digest. Basepair-precise modification of pFL98Q was confirmed by sequencing. In a second Red/ET recombination step, 200 ng of PCR-products 2 or 3 were electroporated into E. coli cells harboring pFL98Q1 and pRed/ET to generate pFL98Q2 and pFL98Q2-stop, respectively. As above, ≥200 ampicillin-, zeocin- and kanamycin-resistant colonies were obtained from the electroporated cultures that had been induced with L-arabinose, while no colonies were obtained from the non-induced controls. Plasmid DNA isolated from individual clones was analyzed by restriction digest and basepair-precise modification of pFL98Q1 was confirmed by sequencing.

After each recombination step, analysis was performed on 6–12 clones. While 100% of the analyzed clones exhibited the expected fragments upon restriction analysis, additional bands indicated that all of the clones harbored mixtures of the target plasmid and the unmodified precursor plasmid (data not shown). This is a common observation when modifying multi-copy plasmids, since usually only very few plasmid copies in a cell undergo recombination. In order to separate recombined and unrecombined plasmid copies, 10 ng of plasmid DNA were retransformed into E. coli HS996. Retransformation clones harbored pure populations of the target plasmid as demonstrated by restriction digest (data not shown).

Plasmids pFL98Q2 and pFL98Q2-stop were subjected to restriction digest with SacII to release the fragments harboring full-length or truncated 98Q-htt cDNA and a downstream FRT-flanked kanamycin resistance cassette, flanked by 50 bp homology arms mediating insertion into the BDNF-BAC. The restriction fragments, 11.4 kb and 2.5 kb in size, were electroporated into E. coli cells harboring BAC RP23–106l19 and pRed/ET. ≥200 chloramphenicol- and kanamycin-resistant clones were obtained from the induced cultures. Twelve clones each were propagated and their BAC DNA was isolated and analyzed for correct integration of the htt cDNA by junction PCR. 100% of the analyzed clones (HTTstop-98Q) or 92% (11 out of 12) of the analyzed clones (FL-HTT-98Q) yielded the correct PCR signals (shown for FL-HTT-98Q clones in supplementary Figure 2A).

With a goal of generating BAC transgenes harboring full-length or truncated htt cDNA with a lower number of Q repeats (15Q) representing the genetic situation in healthy individuals free of HD, BACs RP23–106l19-FL-HTT-98Q and RP23–106l19-HTTstop-98Q were modified by counterselection using the rpsL gene (39). In a first Red/ET recombination step, an rpsL-zeocin counterselection cassette amplified by PCR with primers PCR4–5′ and PCR4–3′ (Supplementary Table 1) was integrated into BACs RP23–106l19-FL-HTT-98Q and RP23–106l19-HTTstop-98Q in place of the 98Q sequence. In a second Red/ET recombination step, the rpsL-zeocin cassette was replaced with a DNA fragment harboring 15Q repeats. The DNA fragment was obtained by PCR amplification from plasmid pIREScDNA15QHtt2, which harbors 15Q htt cDNA. Amplification was performed with primers PCR5–5′ and PCR5–3′ (Supplementary Table 1). After insertion of the rpsL-zeocin counterselection cassette, 12 zeocin-resistant clones each were confirmed by PCR across the junctions of the inserted rpsL-zeo cassette and were further confirmed to exhibit a streptomycin-sensitive phenotype. 100% of the analyzed clones yielded the expected amplification products (data not shown). Following replacement of the rpsL-zeo cassette, 12 streptomycin-resistant clones each were analyzed by PCR across the counterselection site. 100% of the analyzed clones yielded the expected PCR amplicon (shown for FL-HTT-98Q clones in Supplementary Figure 2B).

All four modified BACs were eventually subjected to FLP recombination to remove the kanamycin resistance cassette. BACs RP23–106l19-FL-HTT-98Q-FRT, RP23–106l19-HTTstop-98Q-FRT, RP23–106l19-FL-HTT-15Q-FRT, and RP23–106l19-HTTstop-15Q-FRT were subjected to restriction digest with XhoI followed by pulsed-field gel electrophoresis(PFGE; Figure 2) to confirm BAC integrity. As can be seen from Figure 2, the expected banding pattern was obtained in all cases. All BACs were also confirmed by sequencing across the Q-repeat section and the remaining FRT scar.

Figure 2. Analysis of BAC transgenes by pulsed-field gel-electrophoresis (PFGE). (Click to enlarge)

We have applied Red/ET recombination to generate a set of four BAC transgenes for use in the generation of a novel transgenic mouse model of HD. Expression of full-length 98Q-htt cDNA in 2-week old mice derived from a cross of heterozygous transgenic mice demonstrates the functionality of this BDNF-BAC transgene. Western blotting reveals the presence of the protein in the brainstem (Figure 3), which is a site of endogenous BDNF expression. No cDNA expression was detectable in the cortex, another site of endogenous BDNF expression, since BAC transgenes are subject to insertional effects (40, 41).

Figure 3. Western blot analysis of full-length 98Q htt cDNA expression in mice. (Click to enlarge)

The use of bifunctional homology arms in recombineering protocols has previously been described in specialized recombineering papers dedicated to the modification of large DNA molecules and particularly to BAC fusions (31-35). Bifunctional homology arms were either attached by PCR with chimeric oligonucleotides (31, 32) or were assembled by conventional restriction-ligation techniques (33-35).

Our report illustrates how the concept of chimeric oligonucleotides and bifunctional homology arms can be applied to cloning exercises involving the mobilization of complex cDNAs, such as the generation of BAC transgenes for heterologous expression. The method circumvents PCR amplification of the cDNAs and instead submits their amplification to the E. coli replication machinery. Transgenic mouse lines derived from the BDNF-BAC transgenes generated in this study will add to the repertoire of existing HD mouse models, with the potential to promote a fuller understanding of the molecular mechanisms that govern HD by isolating the cortical contribution to striatal pathophysiology. To our knowledge, this is also the first report describing the transfer of full-length htt cDNA into a heterologous genomic locus.

Competing interests

S.H., S.L., S.N., and H.K. are employees of Gene Bridges GmbH.

2.) Ambrose, C.M., M.P. Duyao, G. Barnes, G.P. Bates, C.S. Lin, J. Srinidhi, S. Baxendale, H. Hummerich. 1994. Structure and expression of the Huntington's disease gene: evidence against simple inactivation due to an expanded CAG repeat. Somat. Cell Mol. Genet. 20:27-38.

3.) Baxendale, S., S. Abdulla, G. Elgar, D. Buck, M. Berks, G. Micklem, R. Durbin, G. Bates. 1995. Comparative sequence analysis of the human and pufferfish Huntington‘s disease genes. Nat. Genet. 10:67-76.

4.) Rubinsztein, D.C., J. Leggo, R. Coles, E. Almqvist, V. Biancalana, J.J. Cassiman, K. Chotai, M. Connarty. 1996. Phenotypic characterization of individuals with 30-40 CAG repeats in the Huntington disease (HD) gene reveals HD cases with 36 repeats and apparently normal elderly individuals with 36-39 repeats. Am. J. Hum. Genet. 59:16-22.

5.) Sathasivam, K., I. Amaechi, L. Mangiarini, and G. Bates. 1997. Identification of an HD patient with a (CAG)180 repeat expansion and the propagation of highly expanded CAG repeats in lambda phage. Hum. Genet. 99:692-695.

6.) Stine, O.C., N. Pleasant, M.L. Franz, M.H. Abbott, S.E. Folstein, and C.A. Ross. 1993. Correlation between the onset age of Huntington's disease and length of the trinucleotide repeat in IT-15. Hum. Mol. Genet. 2:1547-1549.

7.) Wang, L.H., and Z.H. Qin. 2006. Animal models of Huntington‘s disease: implications in uncovering pathogenic mechanisms and developing therapies. Acta Pharmacol. Sin. 27:1287-1302.

8.) Mangiarini, L., K. Sathasivam, M. Seller, B. Cozens, A. Harper, C. Hetherington, M. Lawton, Y. Trottier. 1996. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87:493-506.

9.) Mangiarini, L., K. Sathasivam, A. Mahal, R. Mott, M. Seller, and G.P. Bates. 1997. Instability of highly expanded CAG repeats in mice transgenic for the Huntington's disease mutation. Nat. Genet. 15:197-200.

10.) Schilling, G., M.W. Becher, A.H. Sharp, H.A. Jinnah, K. Duan, J.A. Kotzuk, H.H. Slunt, T. Ratovitski. 1999. Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum. Mol. Genet. 8:397-407.

11.) Gray, M., D.I. Shirasaki, C. Cepeda, V.M. Andre, B. Wilburn, X.H. Lu, J. Tao, I. Yamazaki. 2008. Full-length human mutant huntingtin with a stable polyglutamine repeat can elicit progressive and selective neuropathogenesis in BACHD mice. J. Neurosci. 28:6182-6195.

12.) Slow, E.J., J. van Raamsdonk, D. Rogers, S.H. Coleman, R.K. Graham, Y. Deng, R. Oh, N. Bissada. 2003. Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease. Hum. Mol. Genet. 12:1555-1567.

13.) Hickey, M.A., and M.F. Chesselet. 2003. The use of transgenic and knock-in mice to study Huntington's disease. Cytogenet. Genome Res. 100:276-286.

14.) Shelbourne, P.F., N. Killeen, R.F. Hevner, H.M. Johnston, L. Tecott, M. Lewandoski, M. Ennis, L. Ramirez. 1999. A Huntington's disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice. Hum. Mol. Genet. 8:763-774.

15.) Heng, M.Y., S.J. Tallaksen-Greene, P.J. Detloff, and R.L. Albin. 2007. Longitudinal evaluation of the Hdh(CAG)150 knock-in murine model of Huntington's disease. J. Neurosci. 27:8989-8998.

16.) Muyrers, J.P., Y. Zhang, F. Buchholz, and A.F. Stewart. 2000. RecE/RecT and Redalpha/Redbeta initiate double-stranded break repair by specifically interacting with their respective partners. Genes Dev. 14:1971-1982.

17.) Zhang, Y., F. Buchholz, J.P. Muyrers, and A.F. Stewart. 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20:123-128.

18.) Datsenko, K.A., and B.L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645.

19.) Yu, D., H.M. Ellis, E.C. Lee, N.A. Jenkins, N.G. Copeland, and D.L. Court. 2000. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 97:5978-5983.

20.) Muyrers, J.P., Y. Zhang, G. Testa, and A.F. Stewart. 1999. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27:1555-1557.

21.) Murphy, K.C. 1998. Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J. Bacteriol. 180:2063-2071.

22.) Murphy, K.C., K.G. Campellone, and A.R. Poteete. 2000. PCR-mediated gene replacement in Escherichia coli. Gene 246:321-330.

23.) Radding, C.M., and D.M. Carter. 1971. The role of exonuclease and beta protein of phage lambda in genetic recombination. 3. Binding to deoxyribonucleic acid. J. Biol. Chem. 246:2513-2518.

24.) Kovall, R., and B.W. Matthews. 1997. Toroidal structure of lambda-exonuclease. Science 277:1824-1827.

25.) Kmiec, E., and W.K. Holloman. 1981. Beta protein of bacteriophage lambda promotes renaturation of DNA. J. Biol. Chem. 256:12636-12639.

26.) Muniyappa, K., and C.M. Radding. 1986. The homologous recombination system of phage lambda. Pairing activities of beta protein. J. Biol. Chem. 261:7472-7478.

27.) Murphy, K.C. 1991. Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme. J. Bacteriol. 173:5808-5821.

28.) Zuccato, C., and E. Cattaneo. 2007. Role of brain-derived neurotrophic factor in Huntington's disease. Prog. Neurobiol. 81:294-330.

29.) Koppel, I., T. Aid-Pavlidis, K. Jaanson, M. Sepp, K. Palm, and T. Timmusk. 2010. BAC transgenic mice reveal distal cis-regulatory elements governing BDNF gene expression. Genesis 48:214-219.

30.) Koppel, I., T. Aid-Pavlidis, K. Jaanson, M. Sepp, P. Pruunsild, K. Palm, and T. Timmusk. 2009. Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice. BMC Neurosci. 10:68.

31.) Rivero-Müller, A., S. Lajic, and I. Huhtaniemi. 2007. Assisted large fragment insertion by Red/ET-recombination (ALFIRE)--an alternative and enhanced method for large fragment recombineering. Nucleic Acids Res. 35:e78.

32.) Tischer, B.K., J. von Einem, B. Kaufer, and N. Osterrieder. 2006. Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques 40:191-197.

33.) Sopher, B.L., and A.R. La Spada. 2006. Efficient recombination-based methods for bacterial artificial chromosome fusion and mutagenesis. Gene 371:136-143.

34.) Zhang, X.M., and J.D. Huang. 2003. Combination of overlapping bacterial artificial chromosomes by a two-step recombinogenic engineering method. Nucleic Acids Res. 31:e81.

35.) Maye, P., M.L. Stover, Y. Liu, D.W. Rowe, S. Gong, and A.C. Lichtler. 2009. A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs. BMC Biotechnol. 9:20.

36.) Dower, W.J., J.F. Miller, and C.W. Ragsdale. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16:6127-6145.

37.) Gama-Sosa, M., R. De Gasperi, P.H. Wen, E.A. Gonzalez, K. Kelley, R.A. Lazzarini, and G.A. Elder. 2002. BAC and PAC DNA for the generation of transgenic animals. Biotechniques 33:51-53.

38.) Wang, J., M. Sarov, J. Rientjes, J. Fu, H. Hollak, H. Kranz, W. Xie, A.F. Stewart, and Y. Zhang. 2006. An improved recombineering approach by adding RecA to lambda Red recombination. Mol. Biotechnol. 32:43-53.

39.) Reyrat, J.M., V. Pelicic, B. Gicquel, and R. Rappuoli. 1998. Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect. Immun. 66:4011-4017.

40.) Conner, J.M., J.C. Lauterborn, Q. Yan, C.M. Gall, and S. Varon. 1997. Distribution of brain-derived neurotrophic factor (BDNF) protein and mRNA in the normal adult rat CNS: evidence for anterograde axonal transport. J. Neurosci. 17:2295-2313.

41.) Gong, S., M. Doughty, C.R. Harbaugh, A. Cummins, M.E. Hatten, N. Heintz, and C.R. Gerfen. 2007. Targeting Cre recombinase to specific neuron populations with bacterial artificial chromosome constructs. J. Neurosci. 27:9817-9823.