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Conditional knockout of neural cell adhesion molecule L1 in mouse brain

by Law, Wai-sze

Abstract (Summary)
(Uncorrected OCR) Abstract of thesis entitled

Conditional knockout of neural cell adhesion molecule Ll in mouse brain submitted by

LawWaiSze

for the degree of Master of Philosophy at the University of Hong Kong in Feb 2000

The cell adhesion molecule Ll, which is, predominantly expressed in the developing central and peripheral nervous system, has been implicated in several neuronal processes including neurite outgrowth, neuronal cell migration, neurite fasciculation, myelination, and synaptic plasticity. Main objective of this thesis was to focus on the functional role of Ll in the context of learning and memory. The conditional gene knockout technology using the Cre/loxP site-specific recombination system was used to disrupt L1 function specifically in postnatal mouse brain. First, a Ll gene targeting 10xP vector, pCTLl, was constructed in which the target exon 5 and the pgkneo cassette were flanked by 2 10xP sites. This targeting vector was electroporated into ES cells and selected for ES cell clone with G418. Chimeric male mice were mated with normal C57BL/6J to produce Fl generation, and then FI mice were mated with aCaMKII-Cre transgenic mice to generate brain-specific knockout mice of LI gene. The PCR analysis of genomic DNA isolated from different regions of the mutant brain showed that L 1 gene disruption was restricted to prefrontal cortex and hippocampus. However, Western blot analysis of the expression level of Ll in Ll-floxed mice indicated that Ll protein was absent in the brain homogenate. Northern blot analysis also showed that Ll transcript was absent from the brain. This unexpected result may be due to the presence of the neo resistance gene in the

targeted allele which interfered with the normal splicing of L 1 gene or the pgk promoter driving the neo resistance gene which affected the endogenous promoter activity of Ll. To overcome this problem, another targeting vector was designed in which the pgkneo cassette was flanked by two frt sites. ES cells carrying this mutated allele were transiently transfected with FLP recombinase to remove the pgkneo cassette. Male chimeric mice generated from the FLP-transfected ES cells were mated to C57BU6J females. We now have FI mice that do not carry pgkneo cassette in the targeted allele. After confirming whether these mice will go germline, they will be crossed with aCaMKII-Cre transgenic mice to inactivate Ll in brain subregions. The present data suggest that the optimal orientation of pgkneo, which will not interfere with endogenous expression of gene, appears to be different depends on the gene of interest to delete. The best solution would be to remove the selection markers in ES cells before generating floxed mice.

Bibliographical Information:

Advisor:

School:The University of Hong Kong

School Location:China - Hong Kong SAR

Source Type:Master's Thesis

Keywords:nervous system cell adhesion molecules neuroplasticity mice

ISBN:

Date of Publication:01/01/2000

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