Mutant loxP vectors for selectable marker recycle and conditional knock-outs

BACKGROUND: Gene disruption by targeted integration of transfected constructs becomes increasingly popular for studies of gene function. The chicken B cell line DT40 has been widely used as a model for gene knock-outs due to its high targeted integration activity. Disruption of multiple genes and complementation of the phenotypes is, however, restricted by the number of available selectable marker genes. It is therefore highly desirable to recycle the selectable markers using a site-specific recombination system like Cre/loxP. RESULTS: We constructed three plasmid vectors (neoR, puroR and bsr), which carry selectable marker genes flanked by two different mutant loxP sites. After stable transfection, the marker genes can be excised from the genome by transient induction of Cre recombinase expression. This excision converts the two mutant loxP sites to an inactive double-mutant loxP. Furthermore we constructed a versatile expression vector to clone cDNA expression cassettes between mutant loxP sites. This vector can also be used to design knock-out constructs in which the floxed marker gene is combined with a cDNA expression cassette. This construct enables gene knock-out and complementation in a single step. Gene expression can subsequently be terminated by the Cre mediated deletion of the cDNA expression cassette. This strategy is powerful for analyzing essential genes, whose disruption brings lethality to the mutant cell. CONCLUSIONS: Mutant loxP vectors have been developed for the recycle of selectable markers and conditional gene knock-out approaches. As the marker and the cDNA expression cassettes are driven by the universally active and evolutionary conserved β-actin promoter, they can be used for the selection of stable transfectants in a wide range of cell lines

Early expression of Ig μ chain from a transgene significantly reduces the duration of the pro-B stage but does not affect the small pre-B stage

During B cell development, V-J rearrangements at the Ig heavy μ chain (IgH μ chain) locus occur in early cycling precursors (pro-B stage). Subsequently, rearrangements at the Ig light (IgL) chain locus occur in late resting precursors (small pre-B stage). To study the effects of μ chain expression on the rate of B cell development, purified hematopoletic stem cells (HSC) bearing a μ chain transgene or wild-type HSC were transferred Into Immunodeficlent RAG-2-/-mice and B cell development was followed over time. In addition, cycling B cell precursors were pulse-labeled by the Injection of BrdU into transgenlc and wild-type mice, and the production of BrdU-labeled k+ and λ+ B cells was followed over time. These experiments suggested that early expression of the μ chain from the transgene significantly shortened the duration of the pro-B stage and Immediately drove the precursors to differentiate into small pre-B cells. By contrast, the presence of the transgene did not affect the small pre-B stage, where IgL rearrangements occur. Thus, k and λ rearrangements occurred only after the arrest of cell cycling as previously shown in wild-type mice, even when the μ chain is artificially expressed earlier in B cell developmen

COMPARISON OF THE ACCELERATION PHASE OF SPRINTING BETWEEN COLLEGE SPRINTERS AND COLLEGE BASEBALL PLAYERS

The purpose of this study was to examine and compare the sprinting movement of college sprinters and college baseball players, the latter are assumed to be specialized in the acceleration phase. Twenty subjects, 10 sprinters and 10 baseball players, participated in this study. Lower limbs joint angle (hip, knee and ankle), step length, step frequency, contact time and position of the center of mass at the toe-on and the toe-off were calculated. The results indicated that the center of mass of baseball players was located behind that of sprinters at the 3rd step toe on (p 0.008). In addition, the knee joint was significantly extended at the toe-on in the baseball players at the first and the second steps .These observations indicate that baseball players tend to contact with their foot more forward, and with their knees extended, compared to sprinters

Re-evaluation of the probabilities for productive rearrangements on the κ andλloci

V-J rearrangements at Ig light chain (IgL) genes occur in resting small pre-B cells. In the absence of cell division, the probability of productive κ and λ rearrangements is proportional to the output of κ+ B and λ+ B cells in bone marrow. The kinetics and probability of productive κ or λ rearrangements was assessed in three groups of mice carrying two (wild-type), one or no intact Ig gene, and the following conclusion are drawn,κ and λ rearrangements occur independently at different kinetics, and rearrangements are initiated at a time when κ rearrangements are stopping. The probability of productive κ and λ rearrangements per chromosome is calculated to be −60 and −20% respectively. Thus, a κ gene can attempt rearrangements up to three times per chromosome during B cell development. These findings explain that the observed ratio of κ+ B/λ+ B cell production in wild-type mice is 95/

Occlusal reconstruction of a patient with ameloblastoma ablation using alveolar distraction osteogenesis: a case report

Background Ameloblastoma is one of the most common benign odontogenic neoplasms. Its surgical excision has the potential to lead to postoperative malocclusion. In this case report, we describe the successful interdisciplinary orthodontic treatment of a patient with ameloblastoma who underwent marginal mandibulectomy. Case presentation A woman of 20-year-old was diagnosed with ameloblastoma, and underwent marginal mandibulectomy when she was 8 years of age. She had an excessive overjet (11.5 mm) and a mild open bite (- 1.5 mm) with a severely resorbed atrophic edentulous ridge in the area around the mandibular left lateral incisor, canine and first premolar. An alveolar bone defect associated with tumor resection was regenerated by vertical distraction osteogenesis (DO). Subsequently, 3 dental implants were placed into the reconstructed mandible. Orthodontic treatment using implant-anchored mechanics provided a proper facial profile with significantly improved occlusal function. The occlusion appeared stable for a 7-year retention period. Conclusions These results suggest that surgically assisted and implant anchored-orthodontic approaches might be effective for the correction of such malocclusions

Activation-Induced Cytidine Deaminase Initiates Immunoglobulin Gene Conversion and Hypermutation by a Common Intermediate

Depending on the species and the lymphoid organ, activation-induced cytidine deaminase (AID) expression triggers diversification of the rearranged immunoglobulin (Ig) genes by pseudo V (ψV) gene- templated gene conversion or somatic hypermutation. To investigate how AID can alternatively induce recombination or hypermutation, ψV gene deletions were introduced into the rearranged light chain locus of the DT40 B-cell line. We show that the stepwise removal of the ψV donors not only reduces and eventually abolishes Ig gene conversion, but also activates AID-dependent Ig hypermutation. This strongly supports a model in which AID induces a common modification in the rearranged V(D)J segment, leading to a conversion tract in the presence of nearby donor sequences and to a point mutation in their absence

A New Insight into the Development of Novel Anti-Cancer Drugs that Improve the Expression of Mitochondrial Function-Associated Genes

Recent analyses of the whole genome sequencing data enable us to predict cancer incidence for healthy people at present. In addition, metabolome analyses rediscovered that “cancer is a metabolic disease”. Importantly, it has been suggested that mitochondrial dysfunction might precede the metabolic change. In this chapter, we would discuss if “cancer is a transcriptional disease”. Analyzing 5′-upstream non-protein-encoding regions of the human mitochondrial function-associated genes, we speculate that mitochondrial functions could be recovered or improved at a transcriptional level. In the near future, novel chemo-/gene-therapies might be applied to treat cancer patient converting cancerous cells into normal differentiated cells