Functional analysis of EGFR using a conditional allele

• Main Author: Lee, Tang-Cheng
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2008
• Subjects: Genetics
• ISBN: 0549532617; 9780549532613

Epidermal growth factor receptor (EGFR) is a member of the ERBB family of tyrosine kinase receptors, which includes ERBB2, ERBB3 and ERBB4. EGFR signaling is complicated and involved in a variety of cellular processes, including proliferation, differentiation, migration, adhesion and anti-apoptosis. EGFR has been studied using gene targeting to investigate the in vivo role of the receptor. Constitutional knockout of Egfr in mice results in placental defects with mice dying pre- or perinatally depending on the genetic background. All Egfr null mice that survive to term eventually develop neurodegeneration that is primarily restricted to the cortex and olfactory bulb. Because the existing knockout models are unable to distinguish the cell lineage(s) (i.e. neuron, astrocyte or both) that depend on EGFR signaling for survival, and also result in pre- or peritnatal lethality limiting the ability to study the role of EGFR in physiological functions or diseases of adults in vivo, thus the generation of an Egfr conditional allele is necessary to overcome this limitation. Herein gene targeting was used in combination with the Cre/ loxp system to generate an Egfr conditional allele. This allele was used to develop tissue-specific deletion of EGFR to explore its function in the central nervous system in order to understand the mechanism by which loss of EGFR results in neurodegeneration. After the Egfr conditional allele ( Egfrtm1Dwt) was generated, the Egfr tm1Dwt and EgfrΔ, which is generated by Cre-mediated excision in the Egfrtm1Dwt, were characterized showing that the Egfrtm1Dwt possesses wildtype activity and can be inactivated tissue-specially, and the EgfrΔ is a null allele. To investigate the underlying mechanism responsible for neurodegeneration of Egfr null mice, three Cre transgenic lines expressed in the embryonic epiblast (Sox2-Cre) or neuroprogenitor cells (Nestin-Cre and hGFAP-Cre) were crossed with mice carrying the Egfrtm1Dwt conditional allele to generate Egfrtm1Dwt/tm1Dwt, Sox2-Cre (as well as Nestin-Cre or hGFAP-Cre ) mice. Egfrtm1Dwt/tm1Dwt mice also carrying Sox2-Cre or Nestin-Cre displayed neurodegeneration in the cortex, olfactory bulb and areas surrounding lateral ventricle including corpus callosum, the fimbria of the hippocampus and subventricular zone (SVZ) and rostral extension (RE). These results were consistent with the phenotype of Egfr null mice. Conversely, Egfr tm1Dwt/tm1Dwt, hGFAP-Cre mice showed no damage within the brain. To locate differences in the distribution of Cre expression in the brain that leads to different phenotypes in the brain between Egfrtm1Dwt/tm1Dwt, Nestin-Cre and Egfrtm1Dwt/tm1Dwt, hGFAP-Cre mice, Nestin-Cre and hGFAP-Cre mice were crossed with the ROSA26-lacZ reporter mice (R26R). Results indicated that the hGFAP-Cre line expresses Cre in the progenitors of the cortex, olfactory bulb, SVZ, RE and rostral migratory stream (RMS) but unlike Nestin-Cre not the choroid plexus, despite the lack of neurodegeneration in Egfrtm1Dwt/tm1Dwt, hGFAP-Cre mice. These results suggest that neuronal or astrocytic death in the absence of EGFR signaling is a secondary effect, possibly mediated by the choroid plexus.