Techniques in Recombinant DNA technology DNA重组技术5.ppt

* SO transgenic mice are produced by random integration of a foreign gene into the mouse germ line – foreign DNA is injected into one of the pronuclei ie male and female haploied nuclei contributed by the two parents. This has a good chan * This has a good chance ~10-30% of offsspring will contain foreig DNA in chromosomes of all their tissues and in the germ line Detected by PCR Southern Northern Western. Importantly because the transgene is integrated into the recipient genome by NON HOMOLOGOUS recombination it does not disrupt endogenous genes. * Transgenic mice Mice can be produced with a particular gene inserted at random into the mouse genome. Mice will now contain copies of its own ‘wild-type’ genes PLUS the introduced ‘mutant’ gene copy. This means that the ‘Transgene’ is usually a dominant-negative mutation Example of successfully studied ‘Transgenes’ – 1. Huntington disease Cell specific promoter E.g. neuron specific for Huntingtons Making a transgenic mouse Mutant Gene (e.g. Gln100-Htt) Transcriptional initiation site, introns exons Termination codon and Poly A site Epitope tag- a 10-15 amino acid sequence recognised by a specific monoclonal antibody (e.g. HA1) Need a suitable expression vector Ab res ori Making a transgenic mouse PCR analysis of tail-tip Southern analysis of tissue DNA-notional “copy number” of inserted genes. Northern analysis of tissue mRNA Western analysis of tissue proteins I.e. random insertion by heterologous recombination Transgenic mouse models available – used to understand the process of the disease and can be targeted to specific tissues. SiRNA technology – promising outcomes as potential therapeutic, particularly in dominantly inherited disorders. Location of expression of the mutant gene important for SiRNA strategies – eg specialised area eg brain, skin. * * * So this pro * So this diagram really represents the key steps involved and I hope this will make it clear why it was important to consider all the basic a