A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.     

Concordance analysis for QTL detection in dairy cattle: a case study of leg morphology

Background

The present availability of sequence data gives new opportunities to narrow down from QTL (quantitative trait locus) regions to causative mutations. Our objective was to decrease the number of candidate causative mutations in a QTL region. For this, a concordance analysis was applied for a leg conformation trait in dairy cattle. Several QTL were detected for which the QTL status (homozygous or heterozygous for the QTL) was inferred for each individual. Subsequently, the inferred QTL status was used in a concordance analysis to reduce the number of candidate mutations.

Methods

Twenty QTL for rear leg set side view were mapped using Bayes C. Marker effects estimated during QTL mapping were used to infer the QTL status for each individual. Subsequently, polymorphisms present in the QTL regions were extracted from the whole-genome sequences of 71 Holstein bulls. Only polymorphisms for which the status was concordant with the QTL status were kept as candidate causative mutations.

Results

QTL status could be inferred for 15 of the 20 QTL. The number of concordant polymorphisms differed between QTL and depended on the number of QTL statuses that could be inferred and the linkage disequilibrium in the QTL region. For some QTL, the concordance analysis was efficient and narrowed down to a limited number of candidate mutations located in one or two genes, while for other QTL a large number of genes contained concordant polymorphisms.

Conclusions

For regions for which the concordance analysis could be performed, we were able to reduce the number of candidate mutations. For part of the QTL, the concordant analyses narrowed QTL regions down to a limited number of genes, of which some are known for their role in limb or skeletal development in humans and mice. Mutations in these genes are good candidates for QTN (quantitative trait nucleotides) influencing rear leg set side view.     

The fetal circadian rhythm in pregnancies complicated by pregestational diabetes is altered by maternal glycemic control and the morning cortisol concentration

Circadian rhythmicity is fundamental to human physiology, and is present even during fetal life in normal pregnancies. The impact of maternal endocrine disease on the fetal circadian rhythm is not well understood. The present study aimed to determine the fetal circadian rhythm in pregnancies complicated by pregestational diabetes mellitus (PGDM), compare it with a low-risk reference population, and identify the effects of maternal glycemic control and morning cortisol concentrations. Long-term fetal electrocardiogram recordings were made in 40 women with PGDM at 28 and 36 weeks of gestation. Two recordings were made in 18 of the women (45.0%) and one recording was made in 22 (55.0%). The mean fetal heart rate (fHR) and the fHR variation (root mean square of squared differences) were extracted in 1-min epochs, and circadian rhythmicity was detected by cosinor analysis. The study cohort was divided based on HbA1c levels and morning cortisol concentrations. Statistically, significant circadian rhythms in the fHR and the fHR variation were found in 45 (100%) and 44 (95.7%) of the 45 acceptable PGDM recordings, respectively. The rhythms were similar to those of the reference population. However, there was no statistically significant population-mean rhythm in the fHR among PGDM pregnancies at 36 weeks, indicating an increased interindividual variation. The group with higher HbA1c levels (>6.0%) had no significant population-mean fHR rhythm at 28 or 36 weeks, and no significant fHR-variation rhythm at 36 weeks. Similarly, the group with a lower morning cortisol concentration (≤8.8 µg/dl) had no significant population-mean fHR-variation rhythm at 28 and 36 weeks. These findings indicate that individual fetal rhythmicity is present in pregnancies complicated by PGDM. However, suboptimal maternal glycemic control and a lower maternal morning cortisol concentration are associated with a less-well-synchronized circadian system of the fetus.     

Linkage relationships in the bovine MHC region. High recombination frequency between class II subregions

Class II genes of the bovine major histocompatibility complex (MHC) have been investigated by Southern blot analysis using human DNA probes. Previous studies revealed the presence of bovine DO , DQ , DQ , DR and DR genes, and restriction fragment length polymorphisms for each of these genes were documented. In the present study, the presence of three additional class II genes, designated DZ , DY , and DY , are reported. DZ was assumed to correspond to the human DZ gene while the other two were designated DY because their relationship to human class II genes could not be firmly established. The linkage relationships among bovine class II genes and two additional loci, TCP1B and C4, were investigated by family segregation analysis and analysis of linkage disequilibrium. The results clearly indicated that all these loci belong to the same linkage group. This linkage group is divided into two subregions separated by a fairly high recombination frequency. One region includes the C4, DQ , DQ , DR and DR loci and the other one is composed of the DO DY , DY , and TCPIB loci. No recombinant was observed within any of these subregions and there was a strong or fairly strong linkage disequilibrium between loci within groups. In contrast, as many as five recombinants among three different families were detected in the interval between these subregions giving a recombination frequency estimate of 0.17 ± 0.07. The fairly high recombination frequency observed between class 11 genes in cattle is strikingly different from the corresponding recombination estimates in man and mouse. The finding implies either a much larger molecular distance between some of the bovine class II genes or alternatively the presence of a recombinational hot spot in the bovine class II region.     

The murine receptor for urokinase-type plasminogen activator is primarily expressed in tissues actively undergoing remodeling.

uPAR is a cellular receptor for urokinase plasminogen activator, an enzyme involved in extracellular matrix degradation during processes involving tissue remodeling. We have expressed a recombinant soluble form of murine uPAR and raised rabbit polyclonal antibodies to study the expression of uPAR by immunohistochemistry. The immunohistochemical localization of uPAR was determined in normal mouse organs and in tumors formed by the highly metastatic Lewis lung carcinoma. uPAR immunoreactivity was found in the lungs, kidneys, and spleen, and in endothelial cells in the uterus, urinary bladder, thymus, heart, liver, and testis. No uPAR immunoreactivity was detected in muscle. In general, strong uPAR immunoreactivity was observed in organs undergoing extensive tissue remodeling, as exemplified by trophoblast cells in placenta, and in migrating, but not resting, keratinocytes at the edge of incisional wounds. Staining was not detected in any tissue sections derived from uPAR-deficient mice, thus confirming the specificity of the immunohistochemical staining of uPAR in normal mouse tissues. In Lewis lung carcinoma, uPAR immunoreactivity was found in the tumor cells of the primary tumor and in lung metastases. (J Histochem Cytochem 49:237-246, 2001)     

Binding of a chaperonin to the folding intermediates of lactate dehydrogenase.

When Bacillus stearothermophilus LDH dimer is incubated with increasing concentrations of the denaturant guanidinium chloride, three distinct unfolded states of the molecule are observed at equilibrium [Smith, C. J., et al. (1991) Biochemistry 30, 1028-1036]. The kinetics of LDH refolding are consistent with an unbranched progression through these states. The Escherichia coli chaperonin, GroEL, binds with high affinity to the completely denatured form and more weakly to the earliest folding intermediate, thus retarding the refolding process. A later structurally defined folding intermediate, corresponding to a molten globule form, is not bound by GroEL; neither is the inactive monomer. The complex between GroEL and denatured LDH is destabilized by the binding of magnesium/ATP (Mg/ATP) or by the nonhydrolyzable analogue adenylyl imidodiphosphate (AMP-PNP). From our initial kinetic data, we propose that GroEL exists in two interconvertible forms, one of which is stabilized by the binding of Mg/ATP but associates weakly with the unfolded protein. The other is destabilized by Mg/ATP and associates strongly with unfolded LDH. The relevance of these findings to the role of GroEL in vivo is discussed.     

Biocompatibility of radiolucent breast implants

Current implants for breast augmentation containing silicone gel, saline, or both are radiopaque on mammographic examination and can totally obscure microcalcifications and soft-tissue masses. The effect of these implants on the detection of early breast cancers in patients who have undergone augmentation mammaplasty remains unproven and controversial. Implants filled with medium-chain triglycerides (peanut oil) are radiolucent on mammographic examination and allow visualization of both soft-tissue masses and microcalcifications. To investigate the biocompatibility of radiolucent implants, 10 cc of sterile, nonpyrogenic peanut oil was injected subcutaneously into rats using silicone gel as a control. Twenty-one rabbits had two 125-cc silicone shell implants inserted on either side of the chest wall. The right-sided shell was filled with 125 cc of sterile saline, and the left-sided shell was filled with 125 cc of sterile, nonpyrogenic peanut oil. Results were determined by both histologic and radiographic examination. Rats injected with peanut oil equivalent to 7 percent of their body weight rapidly absorbed the freely injected oil without detriment. Histologic examination of the lungs, liver, kidneys, and tissues adjacent to the injection sites demonstrated no abnormalities. There was no evidence of allergic, toxic, inflammatory, or neoplastic response. Eighteen of 21 rabbits survived more than 3 months. Radiographs showed the oil-filled implants to be radiolucent, whereas the saline-filled controls obscured the surrounding soft and bony tissues. Histologic examination demonstrated a fibrous capsule surrounding both types of implants. Histologic examination of the lungs, liver, and kidneys showed no significant abnormalities. These and previous studies have shown peanut oil to be biocompatible when freely injected either intramuscularly or subcutaneously. This study demonstrates that a radiolucent, peanut oil-filled implant is biocompatible in animals and that further long-term studies for its use in humans are merited.     

NetMHCIIpan-3.0, a common pan-specific MHC class II prediction method including all three human MHC class II isotypes, HLA-DR, HLA-DP and HLA-DQ

Major histocompatibility complex class II (MHCII) molecules play an important role in cell-mediated immunity. They present specific peptides derived from endosomal proteins for recognition by T helper cells. The identification of peptides that bind to MHCII molecules is therefore of great importance for understanding the nature of immune responses and identifying T cell epitopes for the design of new vaccines and immunotherapies. Given the large number of MHC variants, and the costly experimental procedures needed to evaluate individual peptide–MHC interactions, computational predictions have become particularly attractive as first-line methods in epitope discovery. However, only a few so-called pan-specific prediction methods capable of predicting binding to any MHC molecule with known protein sequence are currently available, and all of them are limited to HLA-DR. Here, we present the first pan-specific method capable of predicting peptide binding to any HLA class II molecule with a defined protein sequence. The method employs a strategy common for HLA-DR, HLA-DP and HLA-DQ molecules to define the peptide-binding MHC environment in terms of a pseudo sequence. This strategy allows the inclusion of new molecules even from other species. The method was evaluated in several benchmarks and demonstrates a significant improvement over molecule-specific methods as well as the ability to predict peptide binding of previously uncharacterised MHCII molecules. To the best of our knowledge, the NetMHCIIpan-3.0 method is the first pan-specific predictor covering all HLA class II molecules with known sequences including HLA-DR, HLA-DP, and HLA-DQ. The NetMHCpan-3.0 method is available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.0.     

The same allele of translation initiation factor 4E mediates resistance against two Potyvirus spp. in Pisum sativum

Pathogenicity of two sequenced isolates of Bean yellow mosaic virus (BYMV) was established on genotypes of Pisum sativum L. reported to carry resistance genes to BYMV and other potyviruses. Resistance to the white lupin strain of BYMV (BYMV-W) is inherited as a recessive gene named wlv that maps to linkage group VI together with other Potyvirus resistances. One of these, sbm1, confers resistance to strains of Pea seedborne mosaic virus and previously has been identified as a mutant allele of the eukaryotic translation initiation factor 4E gene (eIF4E). Sequence comparison of eIF4E from BYMV-W-susceptible and -resistant P. sativum genotypes revealed a polymorphism correlating with the resistance profile. Expression of eIF4E from susceptible plants in resistant plants facilitated BYMV-W infection in inoculated leaves. When cDNA of BYMV-W was agroinoculated, resistance mediated by the wlv gene frequently was overcome, and virus from these plants had a codon change causing an Arg to His change at position 116 of the predicted viral genome-linked protein (VPg). Accordingly, plants carrying the wlv resistance gene were infected upon inoculation with BYMV-W derived from cDNA with a His codon at position 116 of the VPg coding region. These results suggested that VPg determined pathogenicity on plants carrying the wlv resistance gene and that wlv corresponded to the sbm1 allele of eIF4E.     

Structure of the Mouse Mammary Tumor Virus: Characterization of Bald Particles

The polypeptide, antigenic, and morphological structure of the mouse mammary tumor virus was studied following protease digestion of intact virions. Intact, untreated virions (rho = 1.17 g/ml) had characteristic envelope spikes, five major polypeptides, and were precipitated by antisera against gp52. Two of the major polypeptides, with molecular weights of 52,000 (gp52) and 36,000 (gp36), had carbohydrate moieties. Protease treatment resulted in spikeless, "bald" particles (rho = 1.14 g/ml), which had altered surface antigenicity and which contained neither gp52 nor gp36. These data indicated that gp52 and gp36 were on the viral envelope. Bald particles retained a 28,000 dalton polypeptide (p28) which was proposed as the major internal polypeptide.