An EYFP insertion mutant containing a modified lox sequence for potential use as a recombination indicator.
The Cre-loxP system has been used for genetic engineering in many organisms ranging from E. coli to mouse. It is known that intermolecular recombination between two loxP sites is substantially less frequent than intramolecular recombination between the same sites. Therefore, for the efficient isolation of intermolecular loxP recombination products, it is essential to develop a powerful screening method. Here, we describe the construction of an EYFP variant (EYFP157) that could become a vital tool in such a screening method. EYFP157 contains, between Gln157 and Lys158, the JT15:JTZ17 mutant loxP sequence that is not cleaved by the Cre protein. When EYFP157 was expressed at 37 degrees C in E. coli and mouse cells, fluorescence of the protein was readily detectable under a standard microscope. EYFP157 possessed 1-3% of the fluorescence from the wild-type EYFP. Interestingly, at room temperature EYFP157 fluoresced more efficiently in E. coli cells and thus the difference in fluorescence intensity between EYFP157 and the wild-type EYFP became negligible. The potential application of EYFP157 as a recombination indicator in both E. coli and mouse cells is further discussed.
Mizutani
Akiko
A
Depertment of Molecular Life Sciences, Division of Basic Molecular Science and Molecular Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan.
Ohtsuka
Masato
M
Kimura
Minoru
M
Tanaka
Masafumi
M
Inoko
Hidetoshi
H
D000595:Amino Acid Sequence; D000818:Animals; D001287:Attachment Sites, Microbiological; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D005819:Genetic Markers; D049452:Green Fluorescent Proteins / Q000235:genetics; D019426:Integrases / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D005819:Genetic Markers; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases