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Molecular Approaches Towards the Isolation of Pediatric Cancer Predisposition Genes
Published in John T. Kemshead, Pediatric Tumors: Immunological and Molecular Markers, 2020
It is conceivable, although unlikely, that these walking strategies can be used to cover the distance from catalase or FSH to the Wilms’ locus (see Figure 12). The large physical distance between even closely linked loci, however, is a major problem. The human haploid genome contains 3 × 109 bp of which, for example, chromosome 11 constitutes 2.4%, i.e., 7.8 × 107 bp. Chromosome band 11p13 (about 4% of the chromosome)contains 3 × 106 bp or 3000 kb. Using cosmid cloning strategies, 45 kb is the largest amount of DNA that can be cloned. To isolate region 11p13, 70 contiguous cosmids would have to be isolated which represents a formidable task even if the libraries are fully representative.106.
A Survey of Newer Gene Probing Techniques
Published in Victor A. Bernstam, Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
Larger cosmid probes designed for marked chromosomal sites (e.g., 11 q 13 and 11 q23 regions) associated with various disease or growth control genes (hematological neoplasias, Ewing’s sarcoma, and ataxiatelangiectasia) can be used in competitive suppression of hybridization to repetitive sequences. This procedure combines biotin-labeled probes and staining of chromosomes with both propidium iodide and 4′,6-diamidino-2-phenylindole (DAPI), producing R and Q banding patterns, respectively. It allows unambiguous chromosome identification of various markers useful in practical analysis of chromosomal alterations.
Vector Technology of Relevance to Nitrogen Fixation Research*
Published in Peter M. Gresshoff, Molecular Biology of Symbiotic Nitrogen Fixation, 2018
Reinhard Simon, Ursula B. Priefer
The cosmid clone identified can also be rescued by transferring the cointegrates back into E. coli where spontaneous resolution occurs; superinfection with λ then separates and purifies the cosmid of interest from the RP4 component.
The light-activated TRP channel: the founding member of the TRP channel superfamily
Published in Journal of Neurogenetics, 2022
However, none of the evidence up to that point provided a definitive answer to the norpA function. The Pak lab, therefore, decided to clone the norpA gene. Deficiency mapping localized the norpA gene to the 4B6–4C1 region of the X chromosome (Banga et al., 1986). A pair of deficiencies whose distal breakpoints were indistinguishable cytologically proved invaluable in the mapping because one of them deleted the norpA function while the other left it intact. The Pak lab obtained from collaborators (Co-authors in (Bloomquist et al., 1988)) a pair of overlapping cosmid clones suspected to cover the mapped region, while in situ hybridization analysis confirmed that the cosmid clones lie within the mapped region. In order to more precisely localize the norpA gene within approx. 50 kb of genomic DNA represented by the cosmid clones, they carried out hybrid dysgenic crosses using the H-E system, in which hobo elements are mobilized (Blackman, Grimaila, Koehler, & Gelbart, 1987), rather than P elements as in the P-M system. They recovered a dysgenic norpA mutant of strong norpA phenotype with a hobo insertion, precisely localizing the norpA site. cDNA clones were isolated using genomic fragments from the hobo insertion and nearby sites and analyzed. Analysis of some of the larger cDNA clones revealed the presence of a single open reading frame, conceptual translation of which would generate a polypeptide of 1095 amino acids.
Tumor regression and immunity in combination therapy with anti-CEA chimeric antigen receptor T cells and anti-CEA-IL2 immunocytokine
Published in OncoImmunology, 2021
Seung E. Cha, Maciej Kujawski, Paul J. Yazaki, Christine Brown, John E. Shively
Mouse care and experimental procedures were performed under pathogen-free conditions in accordance with the Institutional Animal Care and Use Committee of City of Hope (IACUC protocol number 16103). The CEA transgenic mice were previous generated by inserting a 32.6-kb fragment containing the complete human CEA gene and flanking sequences isolated from a genomic cosmid clone and used to produce transgenic C57BL/6 mice as previously described.41 A homozygous line was established that was designated C57BL/6 J-TgN(CEAGe)18FJP. Southern blot analysis showed that this line contained intact copies of the cosmid clone, with approximately 19 integrated copies at one chromosomal location. A mouse-human chimeric anti-CEA monoclonal antibody was used to examine CEA expression by immunohistochemical staining of frozen tissue sections. In the cecum and colon, approximately 20% of the luminal epithelial cells had strong cytoplasmic staining, whereas occasional glands showed intense staining. CEA was also expressed in gastric foveolar cells, whereas small intestine villi had only a few (<1%) positive cells. CEA was not found by immunohistochemistry in other tissues of the digestive tract, nor was it found in a wide range of other tissues or organs.
AggLr, a novel aggregation factor in Lactococcus raffinolactis BGTRK10-1: its role in surface adhesion
Published in Biofouling, 2018
Marija Miljkovic, Pavle Marinkovic, Katarina Novovic, Branko Jovcic, Amarela Terzic-Vidojevic, Milan Kojic
Cosmid DNA (from 350 randomly chosen HB101 clones) was isolated by using the QIAprep Spin Miniprep kit (Qiagen GmBH, Hilden, Germany). The size of the fragments cloned into the cosmid vector was analysed on 1% agarose gel (at a constant voltage of 80 V), after digestion with XbaI. All cosmid derivatives carrying different fragments were transferred into non-aggregating cells (E. faecalis BGZLS10-27 strains) by electroporation (Holo and Nes 1995). Electro-transformation was done with cosmid DNA using an Eppendorf Electroporator (Eppendorf, Hamburg, Germany) set at 25 µF and normally at 2.0 kV. The pulse was delivered using electroporation cuvette (2-mm electrode gap) and exposed to single electrical pulse. Transformants were selected on GM17 Petri dishes containing 10 µg ml−1 of erythromycin after growth for 48 h at 30 °C. Transformants were checked for auto-aggregation ability after overnight growth in the appropriate medium by the auto-aggregation assay. Aggregation was scored as positive when clearly visible snowflake-like particles formed by aggregated cells after vortexing which precipitated and sank to the bottom of the tube leaving a clear supernatant above. For the auto-aggregation assay, the negative control consisted of BGZLS10-27 transformed with the vehicle control pAZILcos vector was used.