Identification of microRNAs specific for high producer CHO cell lines using steady-state cultivation

MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene expression. Hence, microRNAs are considered as potential targets for engineering of Chinese hamster ovary (CHO) cells to improve recombinant protein production. Here, we analyzed and compared the microRNA expression patterns of high, low, and non-producing recombinant CHO cell lines expressing two structurally different model proteins in order to identify microRNAs that are involved in heterologous protein synthesis and secretion and thus might be promising targets for cell engineering to increase productivity. To generate reproducible and comparable data, the cells were cultivated in a bioreactor under steady-state conditions. Global microRNA expression analysis showed that mature microRNAs were predominantly upregulated in the producing cell lines compared to the non-producer. Several microRNAs were significantly differentially expressed between high and low producers, but none of them commonly for both model proteins. The identification of target messenger RNAs (mRNAs) is essential to understand the biological function of microRNAs. Therefore, we negatively correlated microRNA and global mRNA expression data and combined them with computationally predicted and experimentally validated targets. However, statistical analysis of the identified microRNA-mRNA interactions indicated a considerable false positive rate. Our results and the comparison to published data suggest that the reaction of CHO cells to the heterologous protein expression is strongly product- and/or clone-specific. In addition, this study highlights the urgent need for reliable CHO-specific microRNA target prediction tools and experimentally validated target databases in order to facilitate functional analysis of high-throughput microRNA expression data in CHO cells.     

Identification of Repetitive Elements in the Genome of Oreochromis niloticus: Tilapia Repeat Masker

The large-scale bacterial artificial chromosome-end sequencing project of Nile tilapia (Oreochromis niloticus) has generated extensive sequence data that allowed the examination of the repeat content in this fish genome and building of a repeat library specific for this species. This library was established based on Tilapiini repeat sequences from GenBank, sequences orthologous to the repeat library of zebrafish in Repbase, and novel repeats detected by genome analysis using MIRA assembler. We estimate that repeats constitute about 14% of the tilapia genome and also give estimates for the occurrence of the different repeats based on the Basic Local Alignment Search Tool searches within the database of known tilapia sequences. The frequent occurrence of novel repeats in the tilapia genome indicates the importance of using the species-specific repeat masker prior to sequence analyses. A web tool based on the RepeatMasker software was designed to assist tilapia genomics.     

Development of a Cost-Effective Method for Capripoxvirus Genotyping Using Snapback Primer and dsDNA Intercalating Dye

Sheep pox virus (SPPV), goat pox virus (GTPV) and lumpy skin disease virus (LSDV) are very closely related viruses of the Capripoxvirus (CaPV) genus of the Poxviridae family. They are responsible for sheep pox, goat pox and lumpy skin disease which affect sheep, goat and cattle, respectively. The epidemiology of capripox diseases is complex, as some CaPVs are not strictly host-specific. Additionally, the three forms of the disease co-exist in many sub-Saharan countries which complicates the identification of the virus responsible for an outbreak. Genotyping of CaPVs using a low-cost, rapid, highly specific, and easy to perform method allows a swift and accurate identification of the causative agent and significantly assists in selecting appropriate control and eradication measures, such as the most suitable vaccine against the virus during the outbreaks. The objective of this paper is to describe the design and analytical performances of a new molecular assay for CaPV genotyping using unlabelled snapback primers in the presence of dsDNA intercalating EvaGreen dye. This assay was able to simultaneously detect and genotype CaPVs in 63 samples with a sensitivity and specificity of 100%. The genotyping was achieved by observing the melting temperature of snapback stems of the hairpins and those of the full-length amplicons, respectively. Fourteen CaPVs were genotyped as SPPVs, 25 as GTPVs and 24 as LSDVs. The method is highly pathogen specific and cross platform compatible. It is also cost effective as it does not use fluorescently labelled probes, nor require high-resolution melting curve analysis software. Thus it can be easily performed in diagnostic and research laboratories with limited resources. This genotyping method will contribute significantly to the early detection and genotyping of CaPV infection and to epidemiological studies.     

Sequence analysis and characterization of two cryptic plasmids derived from Lactobacillus buchneri CD034

Lactobacillus buchneri is probably the most beneficial microorganism for efficient preservation of animal feed silages made from grass, maize and other plant material against aerobic spoilage. Its obligatory heterofermentative nature, acid resistance and robustness have drawn attention to this species for applications as silage starter culture as well as for genetic engineering. For the first time, two cryptic plasmids present in the same L. buchneri strain, L. buchneri CD034, were isolated, sequenced and characterized. The larger plasmid, designated pCD034-1 was found to be 3424 bp in length with a G + C content of 38.36%. The smaller plasmid, designated pCD034-2 was found to be 2707 bp in length with a G + C content of 38.60%. On both plasmids we predicted three open reading frames. On pCD034-1, ORF 1 encodes a putative replication protein which shares 99% identity with the RepA protein of a Lactobacillus plantarum derived pC194/pUB110-family plasmid. ORF 2 encodes a putative protein of unknown function. ORF 1 and ORF 2 of pCD034-2 correspond to RepA and RepB proteins similar to those of plasmid pLB4 from L. plantarum. ORF 3 of both plasmids encodes a putative mobilization protein similar to that of the pediococcal plasmid pF8801. Double strand origins, putative single strand origins and typical mobilization start signals were identified. Both plasmids were shown to be maintained at relatively high plasmid copy numbers. Two shuttle vectors carrying the origins of replication of pCD034-1 and pCD034-2 were constructed and used to successfully transform two other species isolated from the same environment. Hence, we consider the two novel L. buchneri plasmids a valuable resource for the generation of shuttle and expression vectors for LAB.     

Treatment of Peritoneal Carcinomatosis by Targeted Delivery of the Radio-Labeled Tumor Homing Peptide 213Bi-DTPA-[F3]2 into the Nucleus of Tumor Cells

Background

α-particle emitting isotopes are effective novel tools in cancer therapy, but targeted delivery into tumors is a prerequisite of their application to avoid toxic side effects. Peritoneal carcinomatosis is a widespread dissemination of tumors throughout the peritoneal cavity. As peritoneal carcinomatosis is fatal in most cases, novel therapies are needed. F3 is a tumor homing peptide which is internalized into the nucleus of tumor cells upon binding to nucleolin on the cell surface. Therefore, F3 may be an appropriate carrier for α-particle emitting isotopes facilitating selective tumor therapies.

Principal Findings

A dimer of the vascular tumor homing peptide F3 was chemically coupled to the α-emitter ²¹³Bi (²¹³Bi-DTPA-[F3]₂). We found ²¹³Bi-DTPA-[F3]₂ to accumulate in the nucleus of tumor cells in vitro and in intraperitoneally growing tumors in vivo. To study the anti-tumor activity of ²¹³Bi-DTPA-[F3]₂ we treated mice bearing intraperitoneally growing xenograft tumors with ²¹³Bi-DTPA-[F3]₂. In a tumor prevention study between the days 4–14 after inoculation of tumor cells 6×1.85 MBq (50 µCi) of ²¹³Bi-DTPA-[F3]₂ were injected. In a tumor reduction study between the days 16–26 after inoculation of tumor cells 6×1.85 MBq of ²¹³Bi-DTPA-[F3]₂ were injected. The survival time of the animals was increased from 51 to 93.5 days in the prevention study and from 57 days to 78 days in the tumor reduction study. No toxicity of the treatment was observed. In bio-distribution studies we found ²¹³Bi-DTPA-[F3]₂ to accumulate in tumors but only low activities were found in control organs except for the kidneys, where ²¹³Bi-DTPA-[F3]₂ is found due to renal excretion.

Conclusions/Significance

In conclusion we report that ²¹³Bi-DTPA-[F3]₂ is a novel tool for the targeted delivery of α-emitters into the nucleus of tumor cells that effectively controls peritoneal carcinomatosis in preclinical models and may also be useful in oncology.     

<Emphasis Type="Italic">Trichoplusia ni</Emphasis> cells (High Five<Superscript>TM</Superscript>) are highly efficient for the production of influenza A virus-like particles: a comparison of two insect cell lines as production platforms for influenza vaccines

Virus-like particles (VLPs) consisting of the influenza A virus proteins haemagglutinin (HA) and matrix protein (M1) represent a new alternative approach for vaccine design against influenza virus. Influenza VLPs can be fast and easily produced in sufficient amounts in insect cells using the baculovirus expression system. Up to now, influenza VLPs have been produced in the Spodoptera frugiperda cell line Sf9. We compared VLP production in terms of yield and quality in two insect cell lines, namely Sf9 and the Trichoplusia ni cell line BTI-TN5B1-4 (High Five^TM). Additionally we compared VLP production with three different HAs and two different M1s from influenza H1 and H3 strains including one swine-origin pandemic H1N1 strain. Comparison of the two cell lines showed dramatic differences in baculovirus background as well as in yield and particle density. Taken together, we consider the establishment of the BTI-TN5B1-4 cell line advantageous as production cell line for influenza VLPs.     

Improving baculovirus transduction of mammalian cells by surface display of a RGD-motif

An RGD-containing peptide, comprising 23 amino acids from the foot-and-mouth disease virus (FMDV) VP1 protein was engineered into the envelope of Autographa californica nuclear polyhedrosis virus surface (AcNPV) using two different display strategies. The RGD-motif is a well-described tripeptide, that by binding to cell surface integrins facilitates virus entry into cells. This epitope was displayed, either by directly modifying the native major envelope protein gp64 of AcNPV, or by incorporating a second, modified version of gp64 onto the virus surface. Transduction efficiencies of four mammalian cell lines were compared by detecting the expression of the reporter gene green fluorescent protein (gfp), delivered by the baculovirus genome. Our results showed that insertion of the RGD-peptide into the envelope protein gp64 leads to enhanced specific uptake of baculoviral particles in mammalian cells, only when a combination of wild-type and mutant gp64 was present on the viral surface. Whenever the RGD-peptide was directly inserted into the native gp64, the overall amount of gp64 envelope protein was diminished, leading to decreased viral uptake.