Transcriptome study and identification of potential marker genes related to the stable expression of recombinant proteins in CHO clones

BACKGROUND: Chinese hamster ovary (CHO) cells have become the host of choice for the production of recombinant proteins, due to their capacity for correct protein folding, assembly, and posttranslational modifications. The most widely used system for recombinant proteins is the gene amplification procedure that uses the CHO-Dhfr expression system. However, CHO cells are known to have a very unstable karyotype. This is due to chromosome rearrangements that can arise from translocations and homologous recombination, especially when cells with the CHO-Dhfr expression system are treated with methotrexate hydrate. The present method used in the industry for testing clones for their long-term stability of recombinant protein production is empirical, and it involves their cultivation over extended periods of time prior to the selection of the most suitable clone for further bioprocess development. The aim of the present study was the identification of marker genes that can predict stable expression of recombinant genes in particular clones early in the development stage. RESULTS: The transcriptome profiles of CHO clones with stable and unstable recombinant protein production were investigated over 10-weeks of cultivation, using a DNA microarray. We identified 14 genes that were differentially expressed between the stable and unstable clones already at 2 weeks from the beginning of the cultivation. Their expression was validated by reverse-transcription quantitative real-time PCR (RT-qPCR). Furthermore, the k-nearest neighbour algorithm approach shows that the combination of the gene expression patterns of only five of these 14 genes is sufficient to predict stable recombinant protein production in clones in the early phases of cell-line development. CONCLUSIONS: The exact molecular mechanisms that cause unstable recombinant protein production are not fully understood. However, the expression profiles of some genes in clones with stable and unstable recombinant protein production allow prediction of such instability early in the cell-line development stage. We have thus developed a proof-of-concept for a novel approach to eliminate unstable clones in the CHO-Dhfr expression system, which saves time and labour-intensive work in cell-line development. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12896-015-0218-9) contains supplementary material, which is available to authorized users

Genetic variability within RNA2 of Grapevine fanleaf virus

International audienceThe genetic variability of Grapevine fanleaf virus (GFLV) was assessed within RNA2 of nine isolates from Vitis vinifera cv. Volovnik in a vineyard in Slovenia by immunocapture (IC)-reverse transcription (RT)-polymerase chain reaction (PCR)-restriction length fragment polymorphism (RFLP), followed by cloning and sequencing. Four, one, and nine distinct StyI restrictotypes were identified in the 2AHP, 2BMP, and 2CCP genes, respectively, by IC-RT-PCR-RFLP. Each isolate had a specific StyI RFLP profile across the three RNA2-encoded genes. Sequence analysis of cloned RNA2 ORF amplicons obtained by IC-RT-PCR showed mixed infection in four of the nine isolates and a slightly higher nucleotide variability in the 2AHP and 2CCP genes relative to the 2BMP gene. Also, gene 2AHP, unlike genes 2BMP and 2CCP, had a variable size (765-774 nucleotides) and high amino acid diversity (up to 15%). In addition, a recombination event was identified at nucleotide position 220-225 of gene 2AHP in three of the nine isolates. No clear association was apparent between symptomatology and restrictotype composition, phylogenetic clustering, or occurrence of recombination. This study provides new insights into the genetic diversity of GFLV

Additional file 3: of Transcriptome study and identification of potential marker genes related to the stable expression of recombinant proteins in CHO clones

Names and biological functions of the 14 potential marker genes. Short and full gene names including biological functions of the 14 potential marker genes. (XLSX 13 kb