LectureLecture 24&25 24&25

Embryonic Stem Cell MethodEmbryonic Stem Cell Method

Embryonic Stem Cell Embryonic Stem Cell MethodMethod

• involves prior insertion of the desired involves prior insertion of the desired DNA sequenceDNA sequence

• by homologous recombination into an by homologous recombination into an in vitro culture of embryonic stem (ES) in vitro culture of embryonic stem (ES) cellscells

• ES cells are incorporated into an ES cells are incorporated into an embryo embryo

• at the blastocyst stage of developmentat the blastocyst stage of development

Embryonic Stem Cell Embryonic Stem Cell MethodMethod

• result is a chimeric animalresult is a chimeric animal

• method of choice for gene method of choice for gene inactivation inactivation

• so-called knock-out method so-called knock-out method

What are stem cells?What are stem cells?

• Stem cells are undifferentiated cells Stem cells are undifferentiated cells • have the potential to differentiate into any have the potential to differentiate into any

type of cell (somatic and germ cells) type of cell (somatic and germ cells) • And give rise to a complete organismAnd give rise to a complete organism• harvested from the inner cell mass (ICM) harvested from the inner cell mass (ICM)

of host blastocystsof host blastocysts• grown in culture and retain full potential to grown in culture and retain full potential to

produce all the cells of the mature produce all the cells of the mature individual, including its gametes individual, including its gametes

Establishing transgenic mice with genetically Establishing transgenic mice with genetically engineered ES cellsengineered ES cells

Positive – Negative selectionPositive – Negative selection

Testing for nonspecific integration and Testing for nonspecific integration and homologous recombination in transfected homologous recombination in transfected cells by PCRcells by PCR

Gene disruption by targeted Gene disruption by targeted

homologous recombinanthomologous recombinant