Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs.

Nucleic Acids Research
Marcel WestenbergColin T Dolphin

Abstract

Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a recombinant baculoviral genome, as we wish to engineer the virus into a therapeutically useful gene delivery vector with cell targeting characteristics. Initial attempts to replace gp64 with Fusion (F) genes from other baculoviruses resulted in many rearranged clones in which the counter-selection cassette had been deleted. Bacmid bMON14272 contains nine highly homologous regions (hrs) and deletions were mapped to recombination between hr pairs. Recombineering modifications were attempted to decrease intramolecular recombination and/or increase recombineering efficiency. Of these only the use of longer homology arms on the donor molecule proved effective permitting seamless modification. bMON14272, because of the presence of the hr sequences, can be considered equivalent to a highly repetitive BAC and, as such, the optimized method detailed h...Continue Reading

References

Jan 1, 1988·Annual Review of Microbiology·L K Miller
Oct 15, 1998·Nature Genetics·Y ZhangA F Stewart
Nov 24, 1999·The Journal of General Virology·W F IJkelJ M Vlak
Feb 26, 2000·Gene·B ThyagarajanM P Calos
Oct 5, 2001·Nature Reviews. Genetics·N G CopelandD L Court
Nov 14, 2002·Annual Review of Genetics·Donald L CourtLynn C Thomason
Jan 16, 2003·Nucleic Acids Research·Yuguang ZhaoIan M Jones
Apr 25, 2003·Analytical Biochemistry·Claudia Diaz-CaminoWilliam L Crosby
Mar 17, 2004·Methods in Molecular Biology·Joep P P MuyrersA Francis Stewart
Feb 26, 2005·Nucleic Acids Research·Søren WarmingNeal G Copeland
Jan 27, 2007·Journal of Molecular Biology·Elena MenichelliKiyoshi Nagai
Mar 14, 2008·Current Gene Therapy·Yu-Chen Hu
Jan 23, 2009·Molecular Biotechnology·Shuwen WangJiyue Zhu
Jan 31, 2009·Nature Protocols·Shyam K SharanDonald L Court

❮ Previous
Next ❯

Citations

Dec 27, 2013·Nucleic Acids Research·Hailong WangA Francis Stewart
Aug 4, 2011·Journal of Invertebrate Pathology·Monique M van Oers
Jan 5, 2011·Journal of Biomedicine & Biotechnology·Kumaran Narayanan, Qingwen Chen
Jan 30, 2019·The Journal of General Virology·Bob BoogaardMonique M van Oers

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
PCRs
fluorescence microscopy

Software Mentioned

lox pFinder

Related Concepts

Related Feeds

Artificial Chromosomes

Artificial chromosomes are genetically engineered chromosomes derived from the DNA of a species. Discover the latest research on artificial chromosomes here.