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Animal Models and Cell Culture Systems
In this lecture the educator explains about the Animal Models and Cell Culture Systems. The educator has divided the lecture in 5 sections. In first section she explains about the Inbred Strains and Adoptive Transfer System, SCID Mice and SCID-Human Mice and Cell Culture System.
Inbred Strains is to control experimental variation caused by differences in the genetic backgrounds of experimental animals, immunologists often work with inbred strains that is, genetically identical animals produced by inbreeding.
An autosomal recessive mutation resulting in severe combined immunodeficiency disease (SCID) developed spontaneously in a strain of mice called CB-17. The complexity of the cellular interactions that generate an immune response has led immunologists to rely heavily on various types of in vitro cell-culture systems.
In this section she explains about Radiolabelling Techniques, Biotin Labels, Detection of Small Quantities and Gel Electrophoresis and X-Ray Crystallography. Radioactive labels on antigen or antibody are extremely sensitive markers for detection and quantification. There are many ways to introduce radioactive isotopes into proteins or peptides. In some instances, direct labelling of proteins, especially with enzymes or other large molecules.
When a protein binds to a DNA fragment, forming a DNA protein complex, the electrophoretic mobility of the DNA fragment in a gel is reduced, producing a shift in the position of the band containing that fragment.
Recombinant DNA Technology
In section three she explains about Restriction Enzymes, Cloning Vectors and Cloning of Genomic & cDNA, Polymerase Chain Reaction (PCR) and Blotting. A variety of bacteria produce enzymes, called restriction endonucleases, that degrade foreign DNA(e.g., bacteriophage DNA) but spare the bacterial-cell DNA, which contains methylated residues.
Messenger RNA (mRNA) isolated from cells can be transcribed into complementary DNA (cDNA) with the enzyme reverse transcriptase. The polymerase chain reaction (PCR) is a powerful technique for amplifying specific DNA sequences even when they are present at extremely low levels in a complex mixture.
Gene Transfer to Mammalian Cells
In section four, In Vitro Analysis of Gene Function, In Vivo Analysis of Gene Function, Transgenic Mice and Inducible Genes, Gene Deletion, cre/lox System are discussed. A variety of genes involved in the immune response have been isolated and cloned by use of recombinant DNA techniques. Cloned Genes Transferred into Cultured Cells Allow In vitro Analysis of Gene Function.
Development of techniques to introduce cloned foreign genes (called transgenes) into mouse embryos has permitted immunologists to study the effects of immune-system genes in vivo. In addition to the deletion of genes by gene targeting, recent experimental strategies have been developed that allow the specific deletion of a gene of interest in precisely the tissue of choice.
DNA Regulatory Sequence Analysis and Microarrays
In section five, the educator explains DNA Foot printing, Gel Shift Analysis and CAT Assays and Microarrays, Analysing Patterns of Gene Expression. The transcriptional activity of genes is regulated by promoter and enhancer sequences. These sequences are cis-acting, meaning that they regulate only genes on the same DNA molecule.
When a protein binds to a DNA fragment, forming a DNA protein complex, the electrophoretic mobility of the DNA fragment in a gel is reduced, producing a shift in the position of the band containing that fragment. This phenomenon is the basis of gel-shift analysis.
In the past few years, a new approach has emerged designed to assess differences in gene expression between various cell types or the same cells treated in different fashions. This technology, referred to as microarray technology or gene profiling. The principle is simple and is derived from what we already know about RNA and DNA hybridization. mRNA is isolated from a given sample. Then, when cDNA synthesis is initiated the first strand of the cDNA is labelled with the tag. This forms the pool of target sequences.