Evaluating the influence of selection markers on obtaining selected pools and stable cell lines in human cells

Selection markers are common genetic elements used in recombinant cell line development. While several selection systems exist for use in mammalian cell lines, no previous study has comprehensively evaluated their performance in the isolation of recombinant populations and cell lines. Here we examine four antibiotics, hygromycin B, neomycin, puromycin, and Zeocin™, and their corresponding selector genes, using a green fluorescent protein (GFP) as a reporter in two model cell lines, HT1080 and HEK293. We identify Zeocin™ as the best selection agent for cell line development in human cells. In comparison to the other selection systems, Zeocin™ is able to identify populations with higher fluorescence levels, which in turn leads to the isolation of better clonal populations and less false positives. Furthermore, Zeocin™-resistant populations exhibit better transgene stability in the absence of selection pressure compared to other selection agents. All isolated Zeocin™-resistant clones, regardless of cell type, exhibited GFP expression. By comparison, only 79% of hygromycin B-resistant, 47% of neomycin-resistant, and 14% of puromycin-resistant clones expressed GFP. Based on these results, we rank Zeocin™ > hygromycin B ∼ puromycin > neomycin for cell line development in human cells. Furthermore, this study demonstrates that selection marker choice does indeed impact cell line development.     

The effects of a loin muscling quantitative trait locus (LoinMAX™) on carcass and VIA-based traits in crossbred lambs

LoinMAX (LM) is a quantitative trait locus (QTL), which was found to be segregated in Australian Poll Dorset sheep, and maps to the distal end of sheep chromosome 18. LM-QTL was reported to increase Musculus longissimus dorsi area and weight by 11% and 8%, respectively. The aim of this study was to comprehensively evaluate the direct effects of LM-QTL in a genetic background typical of the stratified structure of the UK sheep industry, before it can be recommended for use in the United Kingdom. Crossbred lambs, either non-carriers or carrying a single copy of LM-QTL, were produced out of Scottish Mule ewes (Bluefaced Leicester × Scottish Blackface) artificially inseminated with semen from two Poll Dorset rams that were heterozygous for LM-QTL. Unexpectedly, one of these rams was also heterozygous for a QTL that affects the overall carcass muscling (MyoMAX™). This was accounted for by nesting MyoMAX™ status (carrier or non-carrier) within sire in the statistical analysis. Lambs were weighed and scanned by using X-ray computed tomography (CT) at an average age of 113 days. Ultrasound scan measurements, along with lamb weights, were taken at an average age of 140 days and lambs were then slaughtered. Carcasses were weighed and classified for fat cover and conformation scores, based on the Meat and Livestock Commission (MLC) carcass classification scheme, and then scanned by using a video image analysis (VIA) system. M. longissimus lumborum (MLL) width, as measured by CT scanning, was greater (P < 0.05) in lambs heterozygous for LM-QTL compared with non-carriers. MLL in LM-QTL carrier lambs was also significantly deeper, as measured by both ultrasound muscle depth at the third lumbar vertebrae (+3.7%; P < 0.05) and CT scanning at the fifth lumbar vertebrae (+3.4%; P < 0.01). Consequently, MLL area, was measured by using CT scanning, was significantly higher (+4.5%; P < 0.01) in lambs carrying a single copy of LM-QTL compared with non-carriers. Additional traits measured by CT, such as leg muscle dimensions, average muscle density and tissue proportions, were not significantly affected by LM-QTL. LM-QTL did not significantly affect total carcass lean or fat weights or MLC conformation and fat score classifications. Using previously derived algorithms, VIA could detect a significant effect of the LM-QTL on the predicted weight of saleable meat yield in the loin primal cut (+2.2%; P < 0.05), but not in the other primal cuts, or the total carcass.     

Modeling human osteosarcoma in mice through 3AB-OS cancer stem cell xenografts

Osteosarcoma is the second leading cause of cancer‐related death for children and young adults. In this study, we have subcutaneously injected—with and without matrigel—athymic mice (Fox1nu/nu) with human osteosarcoma 3AB‐OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB‐OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB‐OS CSC xenografts lacked crucial regulators of beta‐catenin levels (E‐cadherin, APC, and GSK‐3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB‐OS‐derived tumors expressed high levels of pAKT, beta1‐integrin and pFAK, nuclear beta‐catenin, c‐Myc, cyclin D2, along with high levels of hyperphosphorylated‐inactive pRb and anti‐apoptotic proteins such as Bcl‐2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB‐OS tumor xenografts obtained with matrigel co‐injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1‐integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB‐OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1‐integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments. J. Cell. Biochem. 113: 3380–3392, 2012. © 2012 Wiley Periodicals, Inc.     

The physical characterisation of a microscale parallel bioreactor platform with an industrial CHO cell line expressing an IgG4

There is a growing body of evidence that the ambr™ workstation from TAP Biosystems performs well in terms of helping to select appropriate clones for scale-up studies. Here we have investigated the physical characteristics of this microscale bioreactor system and found that these are quite different from those that exist in larger scale stirred bioreactors. For example, the flow regime in the ambr™ vessel is transitional rather than turbulent and the sparged air/oxygen superficial gas velocity is relatively very low whilst the specific power input is much higher (~400 W/m³) when compared to that used at larger scales (typically ~20 W/m³). This specific power input is necessary in order to achieve k_La values sufficiently high to satisfy the oxygen demand of the cells and control of dO₂. In line with other studies, we find that the culture of CHO cells in a 15 mL ambr™ bioreactor gave similar cell growth and productivity to that achieved in a 5 L stirred bioreactor whilst the results from shake flasks were significantly different. Given the differences in physical characteristics between the ambr™ and larger stirred bioreactors, we suggest that this similarity in biological performance is due to their similar control capabilities and the ‘equivalence of the stress parameters’ across the scales when compared with shake flasks.     

Commissioning and performance testing of the first prototype of AlignRT InBore™ a Halcyon™ and Ethos™-dedicated surface guided radiation therapy platform

PurposeTo commission and assess the performance of AlignRT InBore™, a Halcyon™ and Ethos™-dedicated Surface Guided Radiation Therapy (SGRT) platform which combines ceiling-mounted cameras for patient setup and bore-mounted cameras for in-bore tracking.MethodsTo check the potential impact of InBore™ cameras on dose delivery, 16 SRS, H&N, breast and pelvis patients’ quality assurance (QA) treatment plans were measured with/without AlignRT InBore™ and using ArcCHECK® and SRS MapCHECK®. Impact on image quality was determined using Catphan® 540 phantom and considering all available MV and CBCT protocols (head, breast, chest and pelvis). The stability, accuracy and overall performance of AlignRT InBore™ was assessed using an MV Cube and anthropomorphic phantoms.ResultsComparison of 2D dose distributions with/without AlignRT InBore™ showed no impact on treatment delivery for all 16 QA checks (p-value > 0.25). 2D and CBCT images showed no artefacts or change in the contrast-to-noise ratio, resolution and noise values measured with Catphan® 540. Anti-collision sensors were unaffected by the bore-mounted cameras. Additionally, AlignRT InBore™ cameras allowed for motion detection with sub-0.5 mm accuracy and sub-0.4 mm stability with surface coverage of >50 × 60 × 35 cc. Accurate transition (sub-0.3 mm) from virtual to treatment isocentres was achieved. Finally, Halcyon™ rotations during CBCT and beam delivery resulted in limited camera vibrations with translation uncertainty <0.5 mm in left-right and anterior-posterior directions and <0.1 mm in head-feet direction.ConclusionAlignRT InBore™ provides SGRT setup and intrafraction monitoring capabilities with a performance comparable to standard SGRT solutions while having no adverse effect on Halcyon™.     

Microbial community structure of surface sediments from a tropical estuarine environment using next generation sequencing

Microbial community structure was analyzed from tropical monsoon influenced Mandovi-Zuari (Ma-Zu) estuarine sediment by means of Next Gen Sequencing (NGS) approach using Ion Torrent PGM™. The sequencing generated 80,282 raw sequence reads. Barcoding with Ion Tags allowed multiplex analysis of microbial community and helped in identifying shifts in microbial community structure. Analysis of sequence data revealed that sediment at both the stations in the Mandovi estuary was dominated by Archaeal group, Euryarchaeota (53.1% and 64.01%). Among Euryarchaeota, Methanomicrobia was dominant. Methanococci was present only at the mouth and Methanopyri was detected at the mid-estuarine station. Whereas, both the stations of Zuari estuary were dominated by Bacteria, Proteobacteria, mainly Gammaproteobacteria (97.67% and 54.41%). A clear influence of mangrove ecosystem on the bacterial diversity was evident in the Zuari estuary. These results suggest that the two estuaries have a very distinct microbial community structure. Characterization of microbial communities in this study area using NGS for the first time points out that even within geographically close habitats, the microbial population structure is significantly influenced by localized interactions. The signatures obtained from sediments can thus be used to reconstruct habitat characteristics and serve as biomarkers. Future studies should focus on the functional gene profiling of different microbial communities and the influence of seasons and tide in such monsoon influenced estuaries.     

Asymmetric synthesis of (S)-α-(octyl)glycine via alkylation of Ni(II) complex of chiral glycine Schiff base

Over last decade, the use of Ni(II) complexes, derived from of glycine Schiff bases with chiral tridentate ligands, has emerge as a leading methodology for preparation of structurally diverse Tailor-Made Amino Acids, the key structural units in modern medicinal chemistry, and drug design. Here, we report asymmetric synthesis of derivatives of (S)-α-(octyl)glycine ((S)-2-aminodecanoic acid) and its N-Fmoc derivative via alkylation of chiral nucleophilic glycine equivalent with n-octyl bromide. Under the optimized conditions, the alkylation proceeds with excellent yield (98.1%) and diastereoselectivity (98.8% de). The observed stereochemical outcome and convenient reaction conditions bode well for application of this method for large-scale asymmetric synthesis of (S)-2-aminodecanoic acid and its derivatives.     

Large scale purification of linear plasmid DNA for efficient high throughput cloning

In this report we describe a rapid, simple, and efficient method for large-scale purification of linear plasmid DNA to answer demand from high-throughput gene cloning. The process is based on the separation of the linear vector from small DNA fragments by anion exchange chromatography. Gene cloning experiments by restriction/ligation or the In-Fusion(tm) technique confirmed the high quality of the linearized vector as 100% of the genes were successfully cloned.     

Scale-up analysis of geometrically dissimilar single-use bioreactors

Cell culture scale-up is a challenging task due to the simultaneous change of multiple hydrodynamic process characteristics and their different dependencies on the bioreactor size as well as variation in the requirements of individual cell lines. Conventionally, the volumetric power input is the most common parameter to select the impeller speed for scale-up, however, it is well reported that this approach fails when there are huge differences in bioreactor scales. In this study, different scale-up criteria are evaluated. At first, different hydrodynamic characteristics are assessed using computational fluid dynamics data for four single-use bioreactors, the Mobius® CellReady 3 L, the Xcellerex™ XDR-10, the Xcellerex™ XDR-200, and the Xcellerex™ XDR-2000. On the basis of this numerical data, several potential scale-up criteria such as volumetric power input, impeller tip speed, mixing time, maximum hydrodynamic stress, and average strain rate in the impeller zone are evaluated. Out of all these criteria, the latter is found to be most appropriate, and the successful scale-up from 3 to 10 L bioreactor and to 200 L bioreactor is confirmed with cell culture experiments using Chinese Hamster Ovary cell cultivation.