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142 6.2 Generation of the floxedTph2 allele
To follow up the study of the role of 5-HT during both brain development and functioning we decided to generate a Tph2 conditional knockout mouse line in which 5-HT synthesis is abrogated in both a tissue- and time-controlled manner. Such a model will allow the study of the role of 5-HT at specific stages of CNS development.
To this purpose a Tph2 conditional knockout (floxedTph2) allele has been generated via homologous recombination in ES. To generate a floxedTph2 targeting vector, a genomic DNA fragment containing the Tph2 genetic locus was engineered introducing two loxP sites for Cre recombinase-mediated deletion of the Tph2 gene. Since the Tph2 locus spans over 100 kb making unfeasible to flox the entire coding region, the floxedTph2 vector was prepared targeting the 3
rdexon of the Tph2 gene, where a key domain for the Tph2 enzymatic activity is located. The floxedTph2 targeting vector was subsequently introduced into ES cells by electroporation and one clone in which a homologous recombination event had occurred was identified by Southern Blot analysis (Fig 5.1).
Recombinant ES cells thus obtained were then microinjected into C57BL/6 host blastocysts to generate chimeric mice. We microinjected 92 blastocysts, which gave rise to a total number of 27 pups. Among them, 6 showed the typical chimeric patchy coat-colour with cream colour fur derived from the recombinant ES cells and the brown colour fur derived from the host blastocyst, meaning that floxedTph2 knockin ES cells injected contributed to various tissue and organs of the chimeras. Among the chimeric mice generated, two transmitted the floxedTph2 to their progeny, thus allowing the establishment of the floxedTph2 mouse line.
In the floxed Tph2 mouse line the presence of the loxP sites should not
interfere with the normal Tph2 regulation. Conversely, somatic
recombination of the Tph2 conditional allele should result in a Tph2 null
mutation and consequent 5-HT depletion only in those cells where Cre was
activated. FloxedTph2 animals are being mated to transgenic mouse lines
expressing the Cre recombinase in a time- and/or tissue- specific manner. A
peculiar form of Cre recombinase (Cre-ER), inducible by a tamoxifen
treatment, is being used to promote Cre activity at distinct stages of
development. Such a genetic tool will allow abolishing 5-HT synthesis in the
mouse brain from early embryonic to postnatal stages as well as in the adult.
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143
Fig 6.1 Targeting strategy for the generation of conditional knockout floxed Tph2 allele.
Diagram showing the Tph2 locus (a), the targeting vector floxedTph2 (b), and the targeted allele floxedTph2 (c). (d) Southern blot analysis of recombinant floxed Tph2 ES cells. DNA is digested with EcoRI enzyme and probe A external to the left homology arm is used to screen for the 5’ recombination. The expected size of the wild type and recombinant band is 6.2 kb and 4.2 kb respectively. Genomic DNA digested with SpeI is analyzed with probe C external to the right homology arm to screen for the 3’ correct recombination. The expected size of the wild type and recombinant band is 11.5 kb and 4.0 kb respectively. A neo internal probe was also used to exclude the possibility of additional integrations and the size of recombinant band was 16 kb. (e) Giemsa staining of metaphase chromosome of conditional knockout floxed Tph2 ES cells.
