Methods of Evaluation in Orthopaedic Animal Research
Yuehuei H. An, Richard J. Friedman in Animal Models in Orthopaedic Research, 2020
The basic terminology given here is adapted from the reviews by Shore and Kaplan.251,252 A gene is a unit of heredity, consisting of a segment of chromosomal DNA that is required for production of a functional protein or RNA. The gene contains both coding and regulatory regions. A transgene is a foreign gene which has been spliced into an animals original genomic DNA. mRNA is a type of RNA that contains protein coding information. Nucleotide sequence refers to the order of nucleotides in a given segment of DNA or RNA. Translocation is the transfer of a portion of DNA from one chromosome to another. A probe is a DNA or RNA molecule that is labeled, or tagged, and can then be used to locate a complementary DNA or RNA strand through hybridization. Vectors are DNA molecules that are used as carrier molecules for cloned DNA sequences. They contain information which allows recombinant molecules to be replicated in host bacterial cells. A plasmid is a small circular double-stranded DNA molecule which is found in bacteria and replicates independently of the host chromosome. They are commonly used as vectors in molecular cloning. A recombinant DNA molecule is a DNA molecule containing segments of DNA from different origins, such as a piece of human DNA that has been joined to a plasmid DNA. A clone is a term used to describe identical segmental DNA molecules produced by recombinant DNA technique. Molecular cloning is a process by which a specific segment of DNA is isolated and then numerous identical copies, or clones, of that segment of DNA are generated.
Nucleic Acids
Danilo D. Lasic in LIPOSOMES in GENE DELIVERY, 2019
At present, a whole library of these enzymes exists. This field is constantly evolving, particularly to meet the needs of genome mapping. Therefore, in order to isolate larger genomic DNA fragments, restriction enzymes that recognize longer DNA sequence stretches continue to be discovered and made available commercially (currently an enzyme which recognizes a 39-nucleotide-long sequence is available). Restriction enzyme digestion, combined with ligation and purification, can be used to join various genes together as well as to determine the sequence of genes. Figure 3-13 shows schematically the formation of a plasmid. This creates a recombinant plasmid. Such a plasmid normally also contains a gene for resistance against a particular antibiotic which simplifies identification of transfected bacteria. Briefly, these plasmids are introduced into host cells, mostly E. coli bacteria which can replicate it several hundredfold. Bacteria transfected with the correct plasmids are selected upon treatment with a selective agent, such as an antibiotic. Bacteria transfected with the correct plasmids will survive, and after their growth in a bioreactor they are lysed and plasmids are purified. In large-scale preparation large batches of bacteria are used (1 to 100 L) which yield cake (a couple hundred grams per liter broth) which contains 1 to 10 mg of plasmid per liter of bacterial broth. Yields, of course, depend on the size and nature of the plasmid and the strain and harvesting time of the bacteria.
Structural Methods in the Study of Development of the Lung
Joan Gil in Models of Lung Disease, 2020
A cell function expressed in a protein product can now be studied dynamically by determining when the controlling gene is transcribed (deMello et al., 1988). In situ hybridization is a powerful tool that allows posttranscriptional gene expression to be studied within cells in tissue. In situ hybridization (ISH) uses nicktranslated DNA or RNA probes. To make single-stranded DNA probes complementary to specific genes, plasmid DNA containing the gene is nicked, and repaired with radiolabeled nucleotides. The plasmid is then cut and denatured to form single-stranded fragments that include fragments complementary to the gene to be studies. For RNA probes, antisense probes are generated from plasmid vectors. The relevant cDNA fragment is inserted within these vectors in reverse orientation, so that promoter sequences able to transcribe this fragment will be positioned at the far end of the gene fragment, thus transcribing an opposite or antisense strand. In medium containing radiolabeled nucleotides, a specific RNA polymerase promotes the synthesis of labeled antisense RNA. This hybridizes with complementary RNA in fixed frozen or paraffin-wax-embedded tissue sections. Often, ISH and immunohistochemistry (IH) are performed on consecutive adjacent sections to correlate posttranscriptional and posttranslational gene expression.
A pipeline for identification and validation of tumor-specific antigens in a mouse model of metastatic breast cancer
Published in OncoImmunology, 2020
Christa I. DeVette, Harika Gundlapalli, Shu-Chin Alicia Lai, Curtis P. McMurtrey, Ashley R. Hoover, Hem R. Gurung, Wei R. Chen, Alana L. Welm, William H. Hildebrand
The intracellular domain of PyMT (corresponding to amino acids 1–394) and mGM-CSF were cloned into separate pVax1 plasmids (Life Technologies) using restriction digest by EcoRV and HindIII (New England Biolabs)and ligation with T4 DNA ligase (NEB). Plasmid constructs werevalidated by DNA sequencing. pVax1-PyMT and -mGM-CSF plasmids were produced in Top10 cells (Life Technologies) and purified using the PureYield MaxiPrep System (Promega). To confirm protein expression of pVax1-PyMT and pVax1-mGM-CSF in mammalian cells, 293T cells (ATCC) were transiently transfected with Lipofectamine 2000 (Life Technologies) and cell lysates were analyzed by Western blot 24 hours later. Antibodies used include: anti-PyMT (Abcam, ab15085), anti-mGM-CSF (R&D systems, MAB415), and HRP goat anti-rat (Jackson Immunoresearch). Cells were lysed with RIPA buffer containing protease inhibitor cocktails (Roche) then pelleted for protein quantification. Since mGM-CSF is a secreted protein, transfected 293T cells were treated ± the cellular transport inhibitor GolgiPlug (BD Biosciences) for 4 hours prior to analysis.
Determination and identification of antibiotic-resistant oral streptococci isolated from active dental infections in adults
Published in Acta Odontologica Scandinavica, 2018
Juan Pablo Loyola-Rodriguez, Maria Elena Ponce-Diaz, Alejandra Loyola-Leyva, Jose O. Garcia-Cortes, Carlo E. Medina-Solis, Azael A. Contreras-Ramire, Eduardo Serena-Gomez
It is important to notice that in this study, 47.5% of the subjects presented oral streptococci (S. mutans, S. oralis, S. salivarius, S. sanguinis and S. gordonii) from active oral infections that were resistant to antibiotics. The bacterial resistance was mainly observed in clindamycin and moxifloxacin. Bacterial resistance probably depends on the complexity of the bacterial community (biofilm) involved. For example, a study that evaluated a dose–response effect noted that lower antibiotic concentrations showed more bacterial resistance in planktonic bacteria, but dental infections are organized as a biofilm [18]. This microbial biofilms are more tolerant to antibiotic treatment and cause problematic infections through different mechanisms [19,20] such as the transfer of genetic information among bacterial species that provides AR in active dental infections [6]. It has been suggested that this bacterial resistance could be promoted by gene coding, either chromosome or plasmid borne; the difference is that chromosomal DNA is relatively stable, whereas plasmid DNA is easily mobilized from one strain to another bacterial specie [6]. It is possible that ARB could be associated to plasmid-borne since our samples harbor multi-resistant antibiotic bacteria.
TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins
Published in Journal of Receptors and Signal Transduction, 2018
Radka Moravcova, Barbora Melkes, Jiri Novotny
HEK293 cells were transfected with the above-mentioned plasmid using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). Two weeks posttransfection, the cells were sorted on a BD Influx TM cell sorter (BD Biosciences, Franklin Lakes, NJ), and thereupon grown for 6 weeks in the presence of geneticin (800 μg/ml). This resulted in the isolation of several cell clones harboring the introduced plasmid DNA. One of these clones stably expressing TRHR-YFP, designated TRY-1, was used for all subsequent experiments. The expression level of TRHR-YFP in these cells (as assessed by immunoblotting) was comparable to endogenous expression of TRH receptors in pituitary GH1 cells. The specificity of TRHR antibody (Santa Cruz Biotechnology, Santa Cruz, CA; sc-11574) used for this purpose was confirmed elsewhere [14].
Related Knowledge Centers
- Antimicrobial Resistance
- Archaea
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- DNA Construct
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- Genomic DNA
- Molecular Cloning
- Recombinant DNA
- Extrachromosomal DNA
- Vector