Conditional Estrogen Receptor Knockout Mouse Model for Studying Mammary Tumorigenesis
Annual rept. 1 Jan-31 Dec 2002
CINCINNATI UNIV OH
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Toward addressing the functions of ERalpha in breast cancer, we have proposed to conditionally ablate ER-alpha gene in the mammary gland. A gene-targeting vector was generated from a 9.2 KB BamHI fragment containing the ER-alpha exon 3. The construct was confirmed by restriction enzyme digestion and full length DNA sequencing. The linearized targeting vector was electroporated into mouse 129J1 embryonic stem cells ES and G418 neomycin-resistant clones were expanded. Approximately 290 G418-resistant ES clones have been screened for the targeted allele in the mouse genome. Three targeted clones were identified with the 5 and 3 PCR. Southern Blot confirmed two targeted clones. The ES cells harboring the floxed ER-alpha gene have been transfected with an expression vector carrying cre recombinase to delete the Neo gene from the mouse genome of the ES cells. After appropriate selection and characterization, these ES cells will be injected into blastocysts and returned to a pseudo-pregnant host of the same stain for generating the floxed ER-alpha mice. Crossing the homozygous floxed ER-alpha mice with WAP-cre mice is expected to conditionally knockout ER-alpha in lactating mice, allowing us to address many fundamental questions related to estrogen receptor and breast cancer.
- Medicine and Medical Research