Neurotechniques : Neuroscience Gateway

NEUROTECHNIQUES Pacman fever Neuroscience Gateway (December 2006) | doi:10.1038/aba1704

Researchers target the insertion of DNA fragments larger than 100 kb to specific sites in the Drosophila genome.

Pac-Man revolutionized video games. Now, a vector by the same name promises to revolutionize Drosophila genetics. P-element transposons are commonly used in Drosophila transgenesis. But P-element vectors cannot hold large DNA fragments and preferentially insert into regulatory regions of In φC31-mediated integration, genes. Venken et al. used the transgene (top) docks bacterial artificial chromosome (middle) and integrates (BAC) vectors and bacteriophage (bottom) at attP sites in DNA integrase to insert large DNA Drosophila. fragments into the Drosophila genome. They report Drosophila with transgenes as large as 133 kb in a recent article in Science.

The authors created a P-element, C31 artificial chromosome for manipulation (which they call P[acman]), that could hold large DNA fragments. Traditional P elements are high copy-number plasmids, which are ideal for injecting into Drosophila embryos, but become unstable with large DNA inserts. BACs are low copy-number vectors that can hold large fragments of DNA, but are maintained at concentrations too low for transgenesis. Therefore, the authors designed the BAC P[acman] with P-element termini and two different replication origins that could induce either low or high copy number when necessary. To identify transgenic flies, the authors included a white+ marker that makes Drosophila eyes, which are normally red, white.

The larger the DNA insert, the more difficult it is to subclone into a vector because unique restriction enzyme sites are difficult to find in large DNA fragments. Therefore, the authors inserted DNA fragments into P[acman] by 'recombineering', which is based on homologous recombination. They inserted DNA ranging in size from 9.4 to 39 kb into P[acman]. However, Drosophila incorporated only P[acman] vectors with DNA fragments that were smaller than 20 kb. Therefore, P-element transposons could not integrate large DNA fragments into the Drosophila genome.

The bacteriophage C31 inserts its DNA into Streptomyces lividans bacteria. Phage DNA integrates only when bacterial attachment sites (attB) in C31 are in contact with phage attachment sites (attP) in bacteria. The authors placed an attB site into the P[acman] vector. Then they used piggyBac transposons, which are similar to P elements, but insert more randomly into the genome, to create Drosophila lines with attP sites. Therefore, P[acman] should integrate only at attP sites in piggyBac Drosophila. The authors included the yellow + indicator in the piggyBac transposon, so Drosophila that incorporated both attP and P[acman] had yellow bodies and white eyes.

The authors cloned genes, including bancal, Dscam and teashirt, and gene complexes, including Bearded and Enhancer of Spl, as large as 102 kb into P[acman] and expressed them in Drosophila. They used two serial rounds of recombineering to clone one of the largest Drosophila genes, 133 kb Tenascin-major, into P[acman] and successfully expressed it in Drosophila.

BACs are already used to generate transgenic mice carrying not only transgene coding sequences, but their regulatory sequences as well. However, other components of the P[acman] vector might be modified for use in mammals. Bacteriophage integration would help target transgene insertion to limit position effects, by which host sequences adversely regulate transgene expression.

Debra Speert

1. Venken, K. J. T. , He, Y. , Hoskins, R. A. and Bellen, H. J. P[acman]: A BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster . Science 314, 1747–1751 (2006). | Article |