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Published online before printNovember 3, 2015, doi:10.1096/fj.15-273805February 2016The FASEB Journalvol. 30 no. 2 748-760
The major feature of the human pancreatic islet architecture is the organization of endocrine cells into clusters comprising central β cells and peripheral α cells surrounded by vasculature. To have an insight into the mechanisms that govern this unique islet architecture, islet cells were isolated, and reaggregation of α and β cells into islet-like structures (pseudoislets) after culture or transplantation into mice was studied by immunohistology. The pseudoislets formed in culture displayed an unusual cell arrangement, contrasting with the transplanted pseudoislets, which exhibited a cell arrangement similar to that observed in native pancreatic islet subunits. The pattern of revascularization and the distribution of extracellular matrix around transplanted pseudoislets were alike to those observed in native pancreatic islets. This organization of transplanted pseudoislets occurred also when revascularization was abolished by treating mice with an anti-VEGF antibody, but not when contact with extracellular matrix was prevented by encapsulation of pseudoislets within alginate hydrogel. These results indicate that the maintenance of islet cell arrangement is dependent on in vivo features such as extracellular matrix but independent of vascularization.—
Lavallard, V., Armanet, M., Parnaud, G., Meyer, J., Barbieux, C., Montanari, E., Meier, R., Morel, P., Berney, T., Bosco, D. Cell rearrangement in transplanted human islets.
The 7 important molecular tools used in genetic engineering
Gene cloning is the process in which a gene of interest is located and copied out of DNA extracted from an organism. When DNA is extracted from an organism, all of its genes are extracted at one time. This DNA, which contains thousands of different genes. An engineer is a person who constructs and manipulates according to a plan. The term genetic engineer is for an individual who is involved in genetic manipulations.They must find the one specific gene that encodes the specific protein.
The genetic engineer’s molecular tools namely the enzymes most commonly used in genetic engineering experiments are given below:
The discovery of thermostable DNA polymerases, such as Taq Polymerase, made it possible to manipulate DNA replication in the laboratory and was essential to the development of PCR. Primers specific to a particular region of DNA, on either side of the gene of interest, are used, and replication is stopped and started repetitively, generating millions of copies of that gene. These copies can then be separated and purified using gel electrophoresis.
The discovery of enzymes known as restriction endonucleases has been essential to protein engineering. These enzymes cut DNA at specific locations based on the nucleotide sequence. Hundreds of different restriction enzymes, capable of cutting DNA at a distinct site, have been isolated from many different strains of bacteria. DNA cut with a restriction enzyme produces many smaller fragments, of varying sizes. These can be separated using gel electrophoresis or chromatography.
Purifying DNA from a cell culture, or cutting it using restriction enzymes wouldn’t be of much use if we couldn’t visualize the DNA – that is, find a way to view whether or not your extract contains anything, or what size fragments you’ve cut it into. One way to do this is by gel electrophoresis. Gels are used for a variety of purposes, from viewing cut DNA to detecting DNA inserts and knockouts.
In genetic research it is often necessary to link two or more individual strands of DNA, to create a recombinant strand, or close a circular strand that has been cut with restriction enzymes. Enzymes called DNA ligases can create covalent bonds between nucleotide chains. The enzymes DNA polymerase I and polynucleotide kinase are also important in this process, for filling in gaps, or phosphorylating the 5′ ends, respectively.
Small circular pieces of DNA that are not part of a bacterial genome, but are capable of self-replication, are known as plasmids. Plasmids are often used asvectors to transport genes between microorganisms. In biotechnology, once the gene of interest has been amplified and both the gene and plasmid are cut by restriction enzymes, they are ligated together generating what is known as a recombinant DNA. Viral (bacteriophage) DNA can also be used as a vector, as can cosmids, recombinant plasmids containing bacteriophage genes.
The process of transferring genetic material on a vector such as a plasmid, into new host cells, is called transformation. This technique requires that the host cells are exposed to an environmental change which makes them “competent” or temporarily permeable to the vector. Electroporation is one such technique. The larger the plasmid, the lower the efficiency with which it is taken up by cells. Larger DNA segments are more easily cloned using bacteriophage, retrovirus or other viral vectors or cosmids in a method called transduction. Phage or viral vectors are often used in regenerative medicine but may cause insertion of DNA in parts of our chromosomes where we don’t want it, causing complications and even cancer.
Not all cells will take up DNA during transformation. It is essential that there be a method of detecting the ones that do. Generally, plasmids carry genes for antibiotic resistance and transgenic cells can be selected based on expression of those genes and their ability to grow on media containing that antibiotic. Alternative methods of selection depend on the presence of other reporter proteins such as the x-gal/lacZ system, or green fluorescence protein, which allow selection based on color and fluorescence, respectively.
(PDF) الأفكار أو المُعتقدات الخاطئة التي تؤثر سلباً على السيطرة السكرية عند مرضى الداء السكري وكيفية التعامل معها
ماهي الأسباب التي تساهم في زرع المخاوف غير المُبررة علمياً من إستعمال الإنسولين (PDF)
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