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Title: Generation and analysis of ES cells expressing individual isoforms of AML1/Runx1 gene in a constitutive and regulatable manner
Author: Liakhovitskaia, Anna
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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AML1 long splice isoform regulates transcription of many haematopoietic specific genes and may act as both transcriptional activator and repressor. The AML1 short isoform which lacks the transactivation domain can bind DNA but is not capable of activating transcription; suggesting that it acts in a dominant-negative manner. Thus, if the long AML1 isoform induces differentiation of haematopoietic progenitors, the short isoform may promote their self-renewal. This hypothesis may be tested using an engineered ES cell differentiation system overexpressing individual AML1 isoforms. Therefore we aimed here to generate ES cell lines overexpressing individual AML1 isoforms constitutively or in a regulatable fashion in order to test their biological functions. Both, long and short AML1 isoforms demonstrated cytotoxic effect when overexpressed constitutively in ES cells. In order to overcome the toxicity problem, these isoforms have been expressed conditionally in an inducible fashion using the tetracycline gene expression system. Different experimental designs were tried to conditionally overexpress the isoforms in ES cells. Finally, the AML1 isoforms were targeted into the HPRT homing site of Ainv15 ES cells which harbour both regulatory and responsive elements of the tetracycline gene expression system. Since AML1 isoforms were linked to a fluorescent EGFP reporter the expression of AML1 could be readily monitored following induction with doxycycline.  Upon doxycycline induction reduction in size and number of undifferentiated colonies was observed that has not been associated with obvious apoptotic events. AML1 induction has also been initiated during the putative haemangioblast differentiation stage. However, no obvious deviation in clonogenic haematopoietic activity has been observed in induced and non-induced differentiating embryo bodies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available