Nassar, Mohammed A.K.; (1998) Reversible External Control of Gene Expression in an NR1 Knock-in Mouse. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The NMDA type of glutamate receptors plays a crucial role in the induction of long term synaptic potentiation (LTP) which is believed to be one of the molecular basis underlying learning and memory. I have generated a new mouse model for the study of the NMDA receptor function in vivo. This model should allow external (non invasive) reversible control over the native expression of the NR1 gene, the subunit common to all NMDA receptors. My strategy is based on modifying the NR1 gene so that its expression is mediated through the tetracycline-regulated promoter system. This new strategy replaces the NR1 promoter with the PhCMV* promoter and uses the NR1 promoter to drive the expression of the tetracycline-sensitive transactivator (tTA). The NR1 gene was modified in the E14.1 embryonic stem (ES) cell line by homologous recombination with a single targeting vector. A ?22 kb targeting vector was designed and cloned to achieve a tetracycline regulatable expression of NR1 and hence is termed TNR1. Eleven chimeric mice have been produced from 4 of the TNR1-targeted ES clones. One chimera transmitted the ES genome to 7 offspring of which 2 harbour the targeted NR1 allele. Furthermore, a variant of the model was generated where the inserted elements of the tetracycline-regulated promoter system can be deleted in tissues expressing Cre, a site-specific recombinase. As a result, the expression of NR1 gene retains a native tetracycline-independent pattern in Cre expressing tissues. This should allow the control of expression of NR1 in all tissues but those where the loss of NMDA receptor activity causes fatality. A second targeting vector identical to TNR1 but contains two loxP sites, the site for Cre excision, was cloned and is termed TNR1- lox. Nine chimeric mice were obtained from 3 TNR1-lox-targeted ES clones. Two chimeras transmitted the ES genome to 5 offspring of which non harbour the targeted NR1 allele. Breeding of those chimeras is continued to obtained offspring harbouring the TNR1-lox targeted NR1 allele.

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