Single/low-copy integration of transgenes in Caenorhabditis elegans using an ultraviolet trimethylpsoralen method.
BACKGROUND
Transgenic strains of Caenorhabditis elegans are typically generated by injecting DNA into the germline to form multi-copy extrachromosomal arrays. These transgenes are semi-stable and their expression is silenced in the germline. Mos1 transposon or microparticle bombardment methods have been developed to create single- or low-copy chromosomal integrated lines. Here we report an alternative method using ultraviolet trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations.


RESULTS
We successfully integrated low-copy transgenes from extrachromosomal arrays using positive selection based on temperature sensitivity with a vps-45 rescue fragment and negative selection based on benzimidazole sensitivity with a ben-1 rescue fragment. We confirmed that the integrants express transgenes in the germline. Quantitative PCR revealed that strains generated by this method contain single- or low-copy transgenes. Moreover, positive selection marker genes flanked by LoxP sites were excised by Cre recombinase mRNA microinjection, demonstrating Cre-mediated chromosomal excision for the first time in C. elegans.


CONCLUSIONS
Our UV/TMP integration method, based on familiar extrachromosomal transgenics, provides a useful approach for generating single/low-copy gene integrations.
Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan.
Eriko
E
Kage-Nakadai
Hiroyuki
H
Kobuna
Osamu
O
Funatsu
Muneyoshi
M
Otori
Keiko
K
Gengyo-Ando
Sawako
S
Yoshina
Sayaka
S
Hori
Shohei
S
Mitani
D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000235:genetics*; D018628:Gene Dosage; D018014:Gene Transfer Techniques; D019426:Integrases / Q000235:genetics; D014307:Trioxsalen; D014466:Ultraviolet Rays
C045073:Cre recombinase; D019426:Integrases; D014307:Trioxsalen