1. Embryonic Stem Cell Method
Lecture 24&25
Embryonic Stem Cell Method

2. 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

3. Embryonic Stem Cell Method
result is a chimeric animal
method of choice for gene inactivation
so-called knock-out method

4. 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

6. Establishing transgenic mice with genetically engineered ES cells

7. Positive – Negative selection

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

9. Gene disruption by targeted homologous recombinant