EST sequencing for gene discovery in Chinese hamster ovary cells

Abstract

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.

CHO gene expression profiling in biopharmaceutical process analysis and design

“…Harvested cells (2 Â 10 6 cells) were centrifuged and the total RNA was extracted from the cell pellets using the RNeasy Kit (Qiagen GmbH, Hilden, Germany) according to manufacturer's protocol. Ten micrograms of the extracted biotin labeled cRNA was hybridized on a custom-made CHO-specific Affymetrix microarray (Yee et al, 2008b) containing 10,118 probe sets with 7,525 unique sequences that represent a subset from a collection of 16,136 unique CHO cell and Chinese hamster transcripts obtained by comprehensive EST sequencing as described by Wlaschin et al (2005) and Wlaschin and Hu (2007). Samples were scanned in highresolution mode and data were processed with an Affymetrix GeneChip Scanner 3000 system (Affymetrix, Inc., Santa Clara, CA).…”

“…In this study, gene expression profiling was performed in an IgG producing CHO cell line cultivated in industrial fed batch process formats using a CHO-specific Affymetrix microarray (Wlaschin et al, 2005;Yee et al, 2008b) to assess the potential use of this technology in biopharmaceutical process development. Specifically, we investigated the number and extent of gene expression changes during the time course of a representative HT and a LT fed batch process applying two different nutritional regimes.…”

Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

“…Meanwhile, efforts to engineer mouse cells have greatly benefited from numerous genomic tools and technologies, owing in large part to the availability of the Mus musculus reference genome sequence. Genomic resources are also becoming available for CHO cells, such as the CHO-K1 genome 5 , expressed sequence tag 6,7 and bacterial artificial chromosome (BAC) libraries 8 , and compendia of proteomic [9][10][11] and transcriptomic data 7,[12][13][14][15][16] . However, much like how murine cell line data are routinely studied in the context of the Mus musculus reference genome, there is a need for a standard reference for all CHO cell lines to contextualize all of these valuable genomic resources.…”

Development and characterization of a Chinese hamster ovary cell-specific oligonucleotide microarray

“…Until recent years, lack of available sequence from CHO cells has presented a significant barrier to broad spectrum genomics studies. A small number of independent efforts have lead to development of similar tools, most notably the Consortium for CHO Cell Genomics (headed by the laboratory of Wei-Shou Hu at the University of Minnesota, Minneapolis, MN), where sequence mining led to the development of a cDNA microarray platform (Wlaschin et al 2005), and more recently a microarray constructed on the Affymetrix platform (Kantardjieff et al 2009). Several studies in this 3 area have attempted to utilize commercial microarrays from related species, such as mouse, with some success (Ernst et al 2006;Yee et al 2008).…”