EST sequencing for gene discovery in Chinese hamster ovary cells.
Chinese hamster ovary (CHO) cells are one of the most important cell lines in biological research, and are the most widely used host for industrial production of recombinant therapeutic proteins. Despite their extensive applications, little sequence information is available for molecular based research. To facilitate gene discovery and genetic engineering, two cDNA libraries were constructed from three CHO cell lines grown under various conditions. The average insert size for both libraries is approximately 800-850 bp, and each library has comparable redundancy levels of 36%-38% for the sequences isolated. Random sequencing of 4,608 ESTs yielded 2,602 unique assemblies, 76% of which were annotated as orthologs of sequences in the GenBank database. A high abundance of mitochondrial genome transcripts facilitated the assembly of the complete mitochondrial genome by PCR walking. Comparative analysis of sequences from both mitochondrial and nuclear genomes with orthologous genes from other species shows that CHO sequences are generally most similar to mouse; however, examples with highest similarity to rat or human are common. A cDNA microarray, including all 4,608 ESTs, was constructed. The microarray results reveal a high level of consistency between transcript abundance in the libraries and fluorescence intensities. Inclusion of redundant clones in the microarray, additionally, allows small changes in abundant mRNAs to be discerned with a high degree of confidence. The information and tools generated provide access to genomic technology for this important cell line.
Wlaschin
Katie Fraass
KF
University of Minnesota Department of Chemical Engineering and Materials Science, 421 Washington Avenue SE, Minneapolis, Minnesota 55455-0132, USA.
Nissom
Peter Morin
PM
Gatti
Marcela de Leon
Mde L
Ong
Peh Fern
PF
Arleen
Sanny
S
Tan
Kher Shing
KS
Rink
Anette
A
Cham
Breana
B
Wong
Kathy
K
Yap
Miranda
M
Hu
Wei-Shou
WS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002147:Calmodulin / Q000737:chemistry; D006224:Cricetinae; D003412:Cricetulus; D003576:Electron Transport Complex IV / Q000737:chemistry; D020224:Expressed Sequence Tags; D015723:Gene Library; D016678:Genome; D007527:Isoenzymes / Q000737:chemistry; D007770:L-Lactate Dehydrogenase / Q000737:chemistry; D000080864:Lactate Dehydrogenase 5; D008928:Mitochondria / Q000235:genetics; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
C488659:CALM2 protein, human; D002147:Calmodulin; D007527:Isoenzymes; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C040343:ribosomal protein S19; D007770:L-Lactate Dehydrogenase; D000080864:Lactate Dehydrogenase 5; D003576:Electron Transport Complex IV