ABSTRACT
Gene amplification methods play a crucial role in establishment of cells that produce high levels of recombinant protein. However, the stability of such cell lines and the level of recombinant protein produced continue to be suboptimal. Here, we used a combination of a human artificial chromosome (HAC) vector and initiation region (IR)/matrix attachment region (MAR) gene amplification method to establish stable cells that produce high levels of recombinant protein. Amplification of Enhanced green fluorescent protein (EGFP) was induced on a HAC carrying EGFP gene and IR/MAR sequences (EGFP MAR-HAC) in CHO DG44 cells. The expression level of EGFP increased approximately 6-fold compared to the original HAC without IR/MAR sequences. Additionally, anti-vascular endothelial growth factor (VEGF) antibody on a HAC (VEGF MAR-HAC) was also amplified by utilization of this IR/MAR-HAC system, and anti-VEGF antibody levels were approximately 2-fold higher compared with levels in control cells without IR/MAR. Furthermore, the expression of anti-VEGF antibody with VEGF MAR-HAC in CHO-K1 cells increased 2.3-fold compared with that of CHO DG44 cells. Taken together, the IR/MAR-HAC system facilitated amplification of a gene of interest on the HAC vector, and could be used to establish a novel cell line that stably produced protein from mammalian cells.
Subject(s)
Chromosomes, Artificial, Human , Protein Engineering/methods , Recombinant Proteins/genetics , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , CHO Cells , Cricetulus , Genetic Vectors , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Matrix Attachment Regions/genetics , Nucleic Acid Amplification Techniques , Recombinant Proteins/metabolism , Vascular Endothelial Growth Factor A/immunologyABSTRACT
Various Yb²âº-containing fluoride glasses melting under a reductive atmosphere were prepared. The brightest white light emission was observed for an AlF3-based fluoride glass not containing Hf or Zr. The largest full width at half maximum of the white emission spectra was 202 nm. In addition, incorporation of chloride into the AlF3-based glass enabled efficient excitation with near-ultraviolet light corresponding to a GaN bandgap of 3.4 eV and the maximum internal quantum efficiency of Yb²âº: AlF3-based fluoride glass was 42%.
