Improving human interferon-beta production in mammalian cell lines by insertion of an intronic sequence within its naturally uninterrupted gene.
Human beta-interferon is used extensively as a therapeutic agent in a wide variety of diseases, ranging from multiple sclerosis to viral infections. At present, the most common source of interferon-beta is derived from CHO (Chinese-hamster ovary) cells. Interestingly, however, the IFNB gene is characterized by a lack of intronic sequences and therefore does not undergo splicing during its expression pathway. As nuclear processing of pre-mRNA molecules has often been demonstrated to improve production yields of recombinant molecules, we have inserted a heterologous intronic sequence at different positions within the IFNB gene and analysed its effects on protein production. The results obtained in the present study show that the position of intron insertion has profound effects on the expression levels of the IFNB gene and on the nuclear/cytoplasm distribution levels of its mRNA as determined by FISH (fluorescent in situ hybridization) analysis of stably transfected clones. In conclusion, our results provide additional evidence that insertion of intronic sequences may be used to improve protein expression efficiency also in molecules that do not normally undergo any splicing process.
Zago
Paola
P
International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.
Baralle
Marco
M
Ayala
Youhna M
YM
Skoko
Natasa
N
Zacchigna
Serena
S
Buratti
Emanuele
E
Tisminetzky
Sergio
S
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D016899:Interferon-beta / Q000096:biosynthesis* / Q000235:genetics*; D007438:Introns / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D016899:Interferon-beta