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Red/ET recombination with chimeric oligonucleotides allows rapid generation of BAC transgenes harboring full-length or truncated huntingtin cDNA
 
Stefanie Hager1, Saskia Lösch1, Stephan Noll1, Loren Khan-Vaughan2, Michelle E. Ehrlich2, and Harald Kranz1
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Supplementary Material

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.

Acknowledgments

This work was supported by CHDI and NIH NS0045942 and NS052452 to M.E.E. We thank Dr. Marcy MacDonald and Dr. Greg Lawless for the provision of plasmids pFL98Q and pIREScDNA15QHtt2. This paper is subject to the NIH Public Access Policy.

Competing interests

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

Correspondence
Address correspondence to Stefanie Hager, Gene Bridges GmbH, Commercial Centre, Im Neuenheimer Feld 584, 69120 Heidelberg, Germany. Email: [email protected]

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