Transposition Vector Research Articles

MORPHOMETRIC ANALYSIS OF TOPOGRAPHIC VARIABILITY OF THE LEFT AND RIGHT MANDIBULAR CANALS IN CASE OF LOSS OF THE MASTICATORY TEETH

The aim: To study the topographic variability of the left and right mandibular canals in case of bone atrophy caused by the loss of the masticatory teeth. Materials and methods: 136 digital scans were selected for morphometric analysis, 68 for each side taken with the Vatech PaX-i 3D Green extra-oral radiography system. The readout of absolute morphometric values, laying the left and right MC was performed in the projection of 3.7, 3.6, 4.6, 4.7 teeth using standardized Ez3D-I software. Results: The alveolar part is characterized by distance to the alveolar ridge, and primarily exposed to pronounced atrophic processes of bone tissue. Distance to the lingual ridge directly proportionally indicates the morphological transposition vector of the mandibular canals for the distance to the buccal ridge, by the same length to its reduction. Morphometric analysis on a short toothless segment determines the variability of laying the mandibular canals but it is characterized by constant regular values of the ridge of the mandibular base. Conclusions: Dentition defects, moving towards the missing teeth, lead to a decrease in the biophysical stimulus on bone tissue, causing pronounced morphological changes with the loss of significant volume and restructuring of its trabecular layer, which synchronously affects the topographic variability of the left and right MC.

Preventing Elevated Radix Deformity in Asian Rhinoplasty with a Chimeric Dorsal-Glabellar Construct.

Asian facial aesthetic surgery should enhance, but not change, natural features. Augmentation rhinoplasty is a hallmark of Asian cosmetic surgery. In the authors' experience, I-shaped implants can elevate and efface the radix, leading to an unnatural appearance (elevated radix deformity). The Chimeric technique was developed to control final radix position and preserve the nasal profile. We aim to demonstrate that the Chimeric technique promotes forward projection, not elevation, of the radix. Between 2013 and 2015, 49 patients underwent rhinoplasty with I-shaped implants. Nineteen patients had Chimeric dorsal-glabellar implants, 30 did not. Standardized photographs were obtained at every visit. Novel and established photogrammetric parameters were used to describe radix position and position change. A retrospective chart review provided additional procedural details and outcomes data. Patients were followed for 10.8 months (range, 2-36 months). Nasal height increase (113% vs 107%) and bridge length increase (118% vs 105%) were significantly greater when the Chimeric technique was not performed (P < .0001). The nasofrontal angle increased 6° in both groups; there was no difference between groups. The vector of radix position change was 26.1° in the Chimeric group and 63.4° in the traditional group (P < .0001). The Chimeric technique preserves the nasal profile with a favorable (horizontal) radix transposition vector. There was not a significant difference in final radix position when Chimeric rhinoplasty was performed because that is controlled by implant thickness and position. The technique did not blunt the radix significantly. LEVEL OF EVIDENCE 4: Therapeutic.

CLoNe is a new method to target single progenitors and study their progeny in mouse and chick.

Cell lineage analysis enables us to address pivotal questions relating to: the embryonic origin of cells and sibling cell relationships in the adult body; the contribution of progenitors activated after trauma or disease; and the comparison across species in evolutionary biology. To address such fundamental questions, several techniques for clonal labelling have been developed, each with its shortcomings. Here, we report a novel method, CLoNe that is designed to work in all vertebrate species and tissues. CLoNe uses a cocktail of labelling, targeting and transposition vectors that enables targeting of specific subpopulations of progenitor types with a combination of fluorophores resulting in multifluorescence that describes multiple clones per specimen. Furthermore, transposition into the genome ensures the longevity of cell labelling. We demonstrate the robustness of this technique in mouse and chick forebrain development, and show evidence that CLoNe will be broadly applicable to study clonal relationships in different tissues and species.

Development of a high-efficient transformation system of Bacillus pumilus strain DX01 to facilitate gene isolation via gfp-tagged insertional mutagenesis and visualize bacterial colonization of rice roots

A Tn5 transposition vector, pMOD-tet-egfp, was constructed and used for the random insertional mutagenesis of Bacillus pumilus. Various parameters were investigated to increase the transformation efficiency B. pumilus DX01 via Tn5 transposition complexes (transposome): bacterial growth phase, type of electroporation buffer, electric field strength, and recovery medium. Transformation efficiency was up to 3 × 10(4) transformants/μg of DNA under the optimized electroporation conditions, and a total of 1,467 gfp-tagged transformants were obtained. Fluorescence-activated cell sorting analysis showed that all gfp-tagged bacterial cells expressed GFP, indicating that foreign DNA has been successfully integrated into the genome of B. pumilus and expressed. Finally, flanking DNA sequences were isolated from several transformants and colonization of rice roots by B. pumilus DX01 was also studied. The method developed here will be useful for creating an insertion mutant library of gram-positive bacteria, thus facilitating their molecular genetic and cytological studies.

283 GENERATION AND CHARACTERIZATION OF BOVINE INDUCED PLURIPOTENT STEM CELLS

Stem cells in large animals are an excellent model for cell therapy research and fine resources for producing transgenic animals. However, there are only few reports of stem cells in large animals because of technical differences between species. In this report, we successfully generate bovine induced pluripotent stem cells (iPSC) using 4 human reprogramming factors (Oct4, Sox2, Klf4, and c-myc) under control of PiggyBac transposition vector. Fibroblasts derived from bovine fetuses were transfected using FugeneHD agent. After 21 days, colony-shaped structures on the culture plates were mechanically detached and then seeded on a mouse embryonic fibroblast (MEF) feeder layer pretreated with mitomycin C. The culture medium was DMEM/F12 supplemented with 20% serum replacement, 5 ng mL–1 basic fibroblast growth factor (bFGF), 0.1 mM β-mercaptoethanol, 1% NEAA, and 1% penicillin-streptomycin antibiotics. The iPSC colonies showed alkaline phosphatase activity and expressed several pluripotency markers (Oct4, Sox2, SSEA1, and SSEA4). To confirm differentiation potential, the iPSC were cultured as embryoid bodies and then plated again. βIII-tubulin (ectoderm) and GFAP or α-SMA (mesoderm) were well expressed on the attached cells. The results revealed that the bovine fibroblasts were well inducted to iPSC that had potential of multilineage differentiation. We hope this technology contributes to improving transgenic cattle production. This study was financially supported by IPET (grant # 109023-05-3-CG000, 111078-03-1-CG000) and the BK21 program for Veterinary Science.

Putative mannose-specific phosphotransferase system component IID represses expression of suilysin in serotype 2 Streptococcus suis

In this study, we generated a genomic mutant library from a North American strain of serotype 2 Streptococcus suis using the pGh9:IS S1 transposition vector. Suilysin is the hemolysin made by S. suis. A hyper-hemolytic mutant was identified by screening for hemolytic phenotype using media with human blood. The hyper-hemolytic phenotype was characterised by a quantitative hemolysis microplate method. The use of green fluorescent protein (GFP) as a reporter also showed that suilysin gene expression was greater in the mutant. DNA sequence analysis of 3.8 kb surrounding the IS S1 insertion site revealed four open reading frames (ORFs) with three consecutive ORFs that belong to a putative mannose-specific phosphotransferase system (PTS). The S. suis gene homologous to mannose permease IID, manN, was interrupted by the transposon. A complementation test showed that manN repressed the expression of suilysin and the absence of manN was responsible for the hyper-hemolytic phenotype. However, both wild type and isogenic hyper-hemolytic mutant S. suis fermented mannose, glucose and lactose. Thus, despite its potential roles in carbohydrate transport, phosphorylation and metabolism, the manN homologue in the putative mannose-specific PTS regulates gene expression in S. suis.

Integrated physical and genetic mapping of Bacillus cereus and other gram-positive bacteria based on IS231A transposition vectors.

The genome structure of Bacillus cereus is relatively complex, its DNA being modulated between a size-varying chromosome and large plasmids. To study the genetic organization of the B. cereus type strain ATCC 14579, thermosensitive transposition vectors were designed on the basis of IS231A-derived cassettes containing uncommon restriction sites. A highly preferred insertion site for IS231A was detected in the chromosome by Southern blotting and pulsed-field gel electrophoresis (PFGE) analyses of independent insertion mutants. However, once this insertional hot spot was occupied, secondary IS231A insertions occurred randomly, as demonstrated by isolation of independent B. cereus auxotrophs at a frequency of approximately 0.6%. The hot-spot site, as well as several auxotrophic mutations, were mapped by using NotI, SfiI, and AscI PFGE restriction profiles. It was confirmed by sequencing that one of the insertions, generating an Ade- phenotype, had disrupted a gene of the purine synthesis pathway. These results showed that combined PFGE and sequencing analyses of mini-IS231A insertions enable the construction of integrated physical and genetic maps of B. cereus type strain. Moreover, the presence of the ultrarare I-SceI restriction site in the mini-IS231A allowed the isolation, in double-insertion mutants, of contiguous and nonoverlapping large chromosomal fragments, convenient for direct sequencing. The system detailed in this report is therefore a powerful tool for comparative genetic studies among members of the B. cereus group (i.e., B. cereus, B. thuringiensis, B. mycoides, and B. anthracis) and could also be applied to more distantly related gram-positive bacteria.

Characterization of Lactococcus lactis UV-sensitive mutants obtained by ISS1 transposition.

Studies of cellular responses to DNA-damaging agents, mostly in Escherichia coli, have revealed numerous genes and pathways involved in DNA repair. However, other species, particularly those which exist under different environmental conditions than does E. coli, may have rather different responses. Here, we identify and characterize genes involved in DNA repair in a gram-positive plant and dairy bacterium, Lactococcus lactis. Lactococcal strain MG1363 was mutagenized with transposition vector pG+host9::ISS1, and 18 mutants sensitive to mitomycin and UV were isolated at 37 degrees C. DNA sequence analyses allowed the identification of 11 loci and showed that insertions are within genes implicated in DNA metabolism (polA, hexB, and deoB), cell envelope formation (gerC and dltD), various metabolic pathways (arcD, bglA, gidA, hgrP, metB, and proA), and, for seven mutants, nonidentified open reading frames. Seven mutants were chosen for further characterization. They were shown to be UV sensitive at 30 degrees C (the optimal growth temperature of L. lactis); three (gidA, polA, and uvs-75) were affected in their capacity to mediate homologous recombination. Our results indicate that UV resistance of the lactococcal strain can be attributed in part to DNA repair but also suggest that other factors, such as cell envelope composition, may be important in mediating resistance to mutagenic stress.

Transposon mutagenesis of coryneform bacteria.

The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831. The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria. Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 10(4) mutants per microgram DNA. Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants. Auxotrophs could be recovered at a frequency of approximately 0.2%. Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%). No multiple insertions were observed. Chromosomal loci of B. flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria.

Insertion of Transposon Tn 917 Derivatives into the Lactococcus lactis subsp. lactis Chromosome

Two transposition vectors, pTV32 and pLTV1, containing transposon Tn917 derivatives TV32 and LTV1, respectively, were introduced into Lactococcus lactis subsp. lactis MG1614. It was found that pTV32 and pLTV1 replicate and that TV32 and LTV1 transpose in this strain. A protocol for production of a collection of Tn917 insertions in L. lactis subsp. lactis was developed. The physical locations of TV32 on the chromosomal SmaI fragments of 62 independent transpositions were established by pulsed-field gel electrophoresis. These transpositions could be divided into at least 38 different groups that exhibited no Tn917-dominating hot spots on the L. lactis subsp. lactis chromosome. A total of 10 of the 62 transpositions resulted in strains that express beta-galactosidase. This indicates that there was fusion of the promoterless lacZ of the Tn917 derivatives to a chromosomal promoter. Thus, the Tn917-derived transposons should be powerful genetic tools for studying L. lactis subsp. lactis.

New IS 10 transposition vectors based on a Gram-positive replication origin

We describe below a set of plasmid-based vehicles which can be used for delivery of IS 10-derived transposons into Gram − bacteria. These vehicles replicate via a Gram + plasmid origin that is inactive in Escherichia coli; they are easily maintained in Bacillus subtilis. Transposons are introduced by electroporation or transformation with the plasmid, and as in previous delivery systems, transpositions are selected with the appropriate antibiotic. This system should be particularly useful in situations where the standard delivery vehicles, based on bacteriophage λ, are inappropriate. The system described incorporates a number of useful features: a variety of antibiotic markers (Er, Cm, Km or Tc), a polylinker containing restriction sites for rare-cutting endonucleases to facilitate physical mapping of chromosomal insertions, a mutant transposase that confers a relaxation in insertion specificity and positioning of the transposase-encoding gene outside of the transposing segment to ensure the stability of insertions once isolated.

Hsp82-neotransposition vectors to study insertional mutagenesis inDrosophila melanogasterand tissue culture cells

Journal Article Hsp82-neo transposition vectors to study insertional mutagenesis in Drosophila melanogaster and tissue culture cells Get access Heinz Sass Heinz Sass Department of Biochemistry and Molecular Biology, Harvard UniversityCambridge, MA 02138, USA * Present address: Institute of Genetics, Johannes Gutenberg University, PO Box 3980, D-6500 Mainz, FRG Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 17, Issue 24, 25 December 1989, Page 10508, https://doi.org/10.1093/nar/17.24.10508 Published: 25 December 1989 Article history Received: 14 November 1989 Published: 25 December 1989