Targeted Disruption of the Neurotensin Receptor Gene

Gallagher, Edward Jude (1996) Targeted Disruption of the Neurotensin Receptor Gene. PhD thesis, University of Glasgow.

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The primary aim of the work presented in this thesis was to investigate the neurotensin transmission system of the mouse CNS by employing a reverse genetic approach. The neurotensin peptide and receptor distribution within the CNS has been extensively mapped, implicating a role of neurotensin in many brain functions but little is known of the exact functional relationship of neurotensin transmission with respect to other neuronal pathways. However, increasing biochemical, electrophysiological and neuropharmacological data have highlighted a neuromodulatory role of neurotensin over the dopaminergic mesolimbic and nigrostriatal pathways. These pathways are involved in the regulation of movement and motivational behaviour, which are believed to underlie the mechanism of reward reinforcement for such drugs as cocaine, as well as being important to the pathophysiology of schizophrenia. Two gene targeting approaches were undertaken to create a null mutation of the neurotensin high affinity receptor. The first strategy was based on the 'Hit and Run' procedure of Hasty et al., 1992a. An integrative vector was designed based on a 2.8 kb BamHI DNA fragment of the neurotensin receptor gene subcloned from a lambda EMBL3 Balb/c mouse genomic library. Sequence analysis of the BamHI fragment revealed a large open reading frame encoding sequence encompassing the first four transmembrane domains of the receptor. Subsequent vector construction involved the introduction of a mutagenic linker which introduced a stop codon within the sequence encoding the first transmembrane domain of the receptor. PCR analysis of ES cell clones transfected with the integrative vector, (pKLF), indicated the targeted integration of pKLF at the neurotensin receptor gene locus. However, selection of these ES clones for the desired reversion event did not result in the identification of any ES cell clones containing the linker mutation within the NTR gene. Re-evaluation of the 'Hit and Run' technique, in light of the increased understanding of the factors governing gene targeting in ES cells, led to a change in approach. The second strategy utilised a replacement type vector in combination with the 'positive negative selection' regime, (Thomas and Capecchi, 1987), to target the neurotensin receptor gene. The replacement vector used was constructed from a 5' 9.2 kb EcoRI fragment of the neurotensin receptor gene subcloned from the lambda Dash 129J mouse genomic library. The targeting construct introduced a deletion and a disrupting neomycin cassette into the receptor gene within the sequence encoding the fourth transmembrane domain. This targeting construct was successfully used to target a number of ES cell lines; E14, Rl, CGR8 and CGR8.8. Of the 4 targeted Rl ES sublines, 2 produced weakly chimaeric mice, (10-15% ES cell derived). No germline transmission of the null NTR allele was obtained from repeated matings of these chimaeric mice. A targeted CGR8 ES cell subline was used to generate 3 highly chimaeric male mice, (80-90% ES cell derived). However these mice were apparently sterile, with no progeny being produced from repeated matings. Two other targeted sublines were produced from the CGR8.8 ES cell line, and are to be tested for germline transmission. In addition, an in situ hybridisation analysis of the neurotensin receptor expression within the developing mouse embryo, revealed specific localisation of the probe to an unidentified structure of the developing forebrain.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Wayne Davies
Keywords: Genetics
Date of Award: 1996
Depositing User: Enlighten Team
Unique ID: glathesis:1996-75270
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 21:23
Last Modified: 19 Nov 2019 21:23

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