Transformation of Paramecium caudatum with a novel expression vector harboring codon-optimized GFP gene.
We have developed a novel expression vector, pTub-tel3, for transformation in Paramecium caudatum. The vector was constructed by cloning P. caudatum alpha-tubulin 5' and 3' non-coding regions. To examine transformation with the pTub-tel3 construct, we chose the green fluorescent protein (GFP) as a selection marker. When a linearized pTub-tel3 vector containing a GFP open reading frame was injected into the macronucleus, the GFP transcript was expressed in many clones whereas protein expression was detected only after extensive optimization of original GFP codons. GFP-derived fluorescence was distributed throughout the nuclei and cytoplasm except for contractile and food vacuoles. Upon continuous cell division, notable heterogeneity of GFP fluorescence among descendants from the same transformant has emerged. This expression vector can be applied to the analysis of protein trafficking and localization in addition to exogenous gene expression in P. caudatum.
Takenaka
Yasuhiro
Y
Institute of Molecular and Cell Biology (IMCB), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8566, Ibaraki, Japan.
Haga
Nobuyuki
N
Harumoto
Terue
T
Matsuura
Tadashi
T
Mitsui
Youji
Y
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010247:Paramecium / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic; D014404:Tubulin / Q000235:genetics
D008164:Luminescent Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins