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

Iatrogenic lateral meniscus anterior horn injury in different tibial tunnel placement techniques in ACL reconstruction surgery – A cadaveric study

Objectives: The aim of this study was to analyze the effect of tibial tunnel positioning in single bundle and double bundle ACL reconstructions on lateral meniscus anterior root. Materials: Twelve single knee cadavers were used, 6 for a single bundle ACL reconstruction, which were reamed gradually starting from 8 mm, 9 mm and ended with a 10 mm reamers, while the other 6 were prepared for a double bundle ACL reconstruction in which 7 mm reamer for the AM tunnel and 6 mm reamer for the PL tunnel were used. After drilling, changes of lengths and thicknesses of anterior horns of the lateral menisci were recorded. Results: Before drilling, the groups were homogenous for the lateral menisci dimensions. After drilling, no statistically significant difference was noticed between the two groups. However, in single bundle group, 2 anterior horns width injury (1.44 mm and 2.13 mm) with the 9 mm reamer and 3 anterior horns width injury (2.51 mm, 3.55 mm and 4.28 mm) with the 10 mm reamer were recorded. However in double bundle group a single anterior horn width injury (2.82 mm) was recorded. Conclusion: Using a greater size reamer in single bundle reconstruction, causes a relatively higher risk of lateral meniscal anterior root injury. Lateral meniscus stability should be examined arthroscopically after reaming with large reamers. Keywords: Anterior cruciate ligament reconstruction, Anterior meniscus root, Meniscus injury, Tunnel reaming, Cadaver stud