Excision of foreign gene product with cathepsin D in chicken hepatoma cell line.
To easily and rapidly recover exogenous gene products from chicken egg yolk, we constructed pVTG-catD (VTG, vitellogenin; catD, cathepsin D), a vector cassette carrying two catD-recognition signal peptides (catD-RSPs) in addition to the cloning site. An enhanced green fluorescence protein (EGFP)-encoding DNA fragment was ligated into the pVTG-catD. When the resultant construct pVTG-EGFP-catD containing histidine- and myc-tags was transfected into the chicken hepatoma cell line LMH, EGFP-expression at 24h post-cultivation was confirmed by fluorescence microscopy. Because a signal peptide (NTVLAEF) encoded in pVTG-EGFP-catD is recognized by catD, the VTG-EGFP fusion protein digested with catD was detectable by Western blotting. Digested exogenous gene product was recovered with nickel resin. These results indicate that catD-recognition sites bearing pVTG-catD and His-tags are functional in chicken LMH cells. Therefore, the system described here may be of use in making excision exogenous gene products in the chicken and in creating homozygous knock-in chickens.
Sato
Masaharu
M
Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
Kawashima
Tsuyoshi
T
Aosasa
Masayoshi
M
Horiuchi
Hiroyuki
H
Furusawa
Shuichi
S
Matsuda
Haruo
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002402:Cathepsin D / Q000235:genetics*; D045744:Cell Line, Tumor; D002645:Chickens; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000276:immunology; D008114:Liver Neoplasms, Experimental / Q000235:genetics*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data
D017931:DNA Primers; D049452:Green Fluorescent Proteins; D002402:Cathepsin D