BISC439 Lecture Notes - Lecture 2: Northern Blot, Xenopus, Transfection
21 Feb 2017
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Transfected fibroblasts express cadherins aggregate efficiently: transfection: non-viral transduction; put naked dna into a cell by chemical means. Dna is usually a plasmid with a promotor; expresses cdna after promotor. Methods: found similar sequences in chicken n-cad to create xenopus n-cad. Can make in vitro rna from the xenopus cdna to put into the embryo: animal caps dissociated in calcium and magnesium-free medium. Interrupts n-cad and integrin adhesion process: isolated xenopus n-cad cdna, northern analysis & rna protection assays. Used to see if n-cad was in the ectoderm: created rna transcripts by inserting xncad1 & xncad2 into plasmid, experiment 1. Expression of n-cad rna during early frog development. N-cad rna accumulates at neurulation: experiment 2. Tissue distribution of n-cad rna in early embryo. Does not amplify rna so only measures detectable levels. Neural plate & dorsal mesoderm were not cultured. Ectoderm, endoderm, mesoderm, & blastopore lip were cultured to equivalent developmental stage: experiment 3.
