Molecular Cloning, Characterisation, and Gene Targeting Vector Design for the Murine Preprotachykinin-A (PPT-A) and Neurokinin-1 Receptor (NK1-R) Genes

MacKay, Colin Smith (1997) Molecular Cloning, Characterisation, and Gene Targeting Vector Design for the Murine Preprotachykinin-A (PPT-A) and Neurokinin-1 Receptor (NK1-R) Genes. MSc(R) thesis, University of Glasgow.

Abstract

The neuropeptide substance P, recently re-named neurokinin-1 and abbreviated to NK-1, is a member of a family of neuropeptides called the tachykinins. Those neuropeptides are distributed throughout both the mammalian central and peripheral nervous systems and have likely roles in a myriad of cellular functions ranging from nociception to development. Neurokinin-1 has been localised to several interesting regions of the brain, regions that show phenotypic changes in line with the progression of clinical neurodegenerative diseases such as Huntingdons disease for example. NK-1 is encoded by the preprotachykinin-A (PPT-A) gene. The neurokinin-1 receptor (NK-1R) gene encodes the G-protein- coupled receptor for NK-1. In order to investigate the proposed involvement of the PPT-A and NK-1R genes in diseases of the nervous system and other cellular activities, it is important to firstly understand the normal function and regulation of those genes at the molecular level. This project sets out to examine the normal function and regulation of the PPT-A and NK-1R genes by molecularly cloning the murine PPT-A and NK-1R genes and provisionally characterising those genes. Gene targeting vectors for both genes will then be described. Vectors were designed for use in homologous recombination experiments, which is the first step required in order to generate transgenic mice bearing specifically altered forms of those genes. This objective was pursued by firstly cloning the murine NK-1 precursor (PPT-A) gene from a genomic bacteriophage library. A 423 bp probe was generated by polymerase chain reaction (PGR) from mouse genomic DNA template and was subcloned and sequenced. Optimal hybridisation conditions were then determined and the probe used to isolate lambda bacteriophage clones from a library. Similarly, positively-hybridising clones of the NK-1R (receptor) gene were isolated from another bacteriophage library using an 865 bp rat NK-1 R cDNA-derived probe. Secondly, the PPT-A bacteriophage clones were analysed by restriction mapping, PCR and by hybridisation to exon-specific probes. The exons of the murine PPT-A gene were then subcloned, restriction mapped and positioned relative to each other on linear restriction maps. Some mouse PPT-A sequence was obtained and aligned with other species to determine the extent of identity and homology. A similar approach was used for the NK-1R bacteriophage clones. Thirdly, gene targeting vectors for use in homologous recombination experiments in mouse embryonic stem cells were then constructed for each of the genes. Multiple cloning and subcloning steps were required to do this. Several endonuclease recognition sequences were altered by ligation of DNA linker sequences. A neomycin (G418) resistance gene with its own promoter was introduced into PPT-A exon 3, or in the the other vector, the neomycin resistance gene was introduced in place of a deleted 2.4 kb genomic sequence containing PPT-A exons 3 and 4. For the NK-1R vector, exons 3 and 4 were deleted and replaced by the neo resistance gene. In each vector, a thymidine kinase (TK) counter-selection gene from herpes simplex I virus (HSV1) was also inserted.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Additional Information: Adviser: Davies Wayne
• Keywords: Genetics
• Date of Award: 1997
• Depositing User: Enlighten Team
• Unique ID: glathesis:1997-76211
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 16:28
• Last Modified: 19 Nov 2019 16:28
• URI: https://theses.gla.ac.uk/id/eprint/76211