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Lecture 24&25 Embryonic Stem Cell Method Embryonic Stem Cell Method • involves prior insertion of the desired DNA sequence • by homologous recombination into an in vitro culture of embryonic stem (ES) cells • ES cells are incorporated into an embryo • at the blastocyst stage of development Embryonic Stem Cell Method • result is a chimeric animal • method of choice for gene inactivation • so-called knock-out method What are stem cells? • Stem cells are undifferentiated cells • have the potential to differentiate into any type • • • of cell (somatic and germ cells) And give rise to a complete organism harvested from the inner cell mass (ICM) of host blastocysts grown in culture and retain full potential to produce all the cells of the mature individual, including its gametes Establishing transgenic mice with genetically engineered ES cells Positive – Negative selection Testing for nonspecific integration and homologous recombination in transfected cells by PCR Gene disruption by targeted homologous recombinant