2Mount Sinai Medical Center, Annenberg Building, New York, NY, USA
Our study demonstrates how a recombineering protocol involving bifunctional homology arms and chimeric oligonucleotides can be applied to the mobilization of a complex cDNA. We have applied this method to transfer the notoriously difficult huntingtin cDNA into a heterologous genomic locus and have thus generated a set of novel BAC transgenes which will further research into Huntington's disease.
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.