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.     

FTA-DNA直接提取法在病原真菌分子鉴定中的应用

目的建立并评价FTA-DNA直接提取法在病原真菌分子鉴定中的应用。方法采用whatman FTA-DNA直接提取法从25个不同种属的45株培养的菌株和6例临床标本中提取病原真菌DNA,用于病原真菌的测序鉴定。配制不同浓度的孢子悬液探索该方法的检测限和安全性。结果 45株菌株扩增后均能得到1条清晰的DNA扩增片段,并成功测序。应用该方法亦成功从腹水、胸水、口腔拭子、宫颈拭子来源的临床标本中直接提取DNA并成功鉴定病原真菌。该DNA提取方法联合降落PCR能检测到1.0×103个cell/mL的孢子悬液,1.0×104个cell/mL及以下浓度的孢子悬液可以被FTA卡完全灭活。结论 FTA-DNA直接提取法可快速有效地从培养的菌株及部分临床标本中提取并保存病原真菌DNA,用于病原真菌的测序鉴定。     

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.     

FTA卡和普通定性滤纸提取DNA方法研究

用FTA采样卡和普通定性滤纸采集藏绵羊血样,采用NaOH法提取血液基因组DNA,利用设计的一对引物对DRB1基因第三外显子进行扩增,通过PCR产物琼脂糖凝胶检测,对普通定性滤纸与FTA采样卡的两种提取DNA的方法进行比较,结果认为采用普通定性滤纸-NaOH法提取血液基因组DNA,NaOH的最佳洗涤浓度是25 mmol/L,采用FTA-NaOH法提取血液基因组DNA,NaOH的最佳洗涤浓度是20 mmol/L,但普通定性滤纸法提取血液基因组DNA平均成本远低于FTA采样卡,普通定性滤纸法提取血液基因组DNA具有快速、便捷、经济及高效的特点.     

Polymorphic microsatellite markers for Philippine tarsiers (<Emphasis Type="Italic">Tarsius syrichta</Emphasis>)

This study reports the isolation and characterization of eight microsatellite markers for the study of Philippine tarsiers (Tarsius syrichta), small primates endemic to this Southeast Asian archipelago. The markers were used to screen 14 Tarsius syrichta for allelic diversity. This suite of highly polymorphic microsatellites provides the first chance to genetically study parentage and dispersal patterns in Philippine tarsiers.     

Simple,filter-based PCR detection of Thelohania solenopsae (Microspora) in fire ants (Solenopsis invicta)

Thelohania solenopsae is a microsporidian parasite that may serve as a biological control agent for the red imported fire ant, Solenopsis invicta. A rapid, filter-based PCR amplification method detecting a portion of the small-subunit ribosomal RNA gene was developed to facilitate field studies detecting the parasite in fire ants. Processing ant homogenates with a commercially available membrane-based system, FTA Classic Card technology, compared favorably with traditional DNA extraction and PCR amplification methods. As few as 100 spores were detected. The FTA membrane system is a simple, extraction-free method for detecting T. solenopsae in fire ants, and allows for easy archival storage of DNA samples.     

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™.     

Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms

We present two methods for DNA extraction from fresh and dried mushrooms that are adaptable to high-throughput sequencing initiatives, such as DNA barcoding. Our results show that these protocols yield ∼85% sequencing success from recently collected materials. Tests with both recent (<2 year) and older (>100 years) specimens reveal that older collections have low success rates and may be an inefficient resource for populating a barcode database. However, our method of extracting DNA from herbarium samples using small amount of tissue is reliable and could be used for important historical specimens. The application of these protocols greatly reduces time, and therefore cost, of generating DNA sequences from mushrooms and other fungi vs. traditional extraction methods. The efficiency of these methods illustrates that standardization and streamlining of sample processing should be shifted from the laboratory to the field.