A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.
Mosaic Analysis with Double Markers technology is a powerful method that can provide highly quantitative information about the proliferation behavior and cellar outputs of individuals stem cells.A unique feature of this technique, is that it permits lineage tracing and functional genetic analysis of candidate genes at the Single Cell level.demontrating to procedure will be, Carmen Streicher, your technician, Nicole Amberge, post-doc, Gisselle Cheung, post-doc and Andy Hansen, a PhD student from my laboratory.To induce MADM recombination events in experimental Mice, use a 1ml syringe equipped with a 25 gauge needle to deliver a single injection of Tamoxifen Intraperitoneal into a timed to pregnant dam.From MADM clonal analysis to postnatal time points.On embryonic days 18 to 19, place a pregnant mouse in the supine position and disinfect the fur with 70%ethanol.Make a small incision in the skin in the lower abdomen above the uterus and make a second incision through the muscles and the abdominal muscular wall to reveal the peritoneum.Use scissors to separate the uterus from the surrounding tissues and place the intact uterus into a glove with warm water.Then use fine tip scissors and fingers to carefully open the uterine walls to release the embryos.Clean the pups gently pressing the chests from time to time to initiate breathing before transferring the prenatal animals to a foster mother.To collect neural MADM Clone Tissue place an anesthetized animal in the supine position on a perfusion surgery tray and disinfect the fur with 70%ethanol.Use scissors and forceps to make one incision through the skin and one incision through the muscle layer.When the thoracic cavity is visible, sniff and lift the diaphragm to reveal the heart and carefully trim and pin the rib cage to the surgical tray.insert a needle connected to a peristaltic pump tubing filled with PBS into the lower left ventricle of the heart and use small Iris scissors to make an incision in the posterior end of the right atrium.Perfuse the cardiovascular system with the PBS followed immediately by perfusion with freshly prepared ice cold 4%paraformaldehyde in PBS.When the perfusion is complete, remove the brain through careful dissection and place the harvested organ in at least five times the brain volume of fresh 4%paraformaldehyde overnight at 4
