SAFER: Transgenic mice as innovative systems for the screening and the pharmacokinetic analysis of new molecules active through nuclear receptors

• Unité de recherche: PCRD EU
• Numéro de projet: 97.0001
• Titre du projet anglais: SAFER: Transgenic mice as innovative systems for the screening and the pharmacokinetic analysis of new molecules active through nuclear receptors

Mots-clé (Anglais)

Transcription; gene expression; reporter assay; in situ; transgenic; pharmacokinetics; estrogens; beta lactamase

Autre Numéro de projet (Anglais)

EU project number: BMH4-CT97-2286

Programme de recherche (Anglais)

EU-programme: 4. Frame Research Programme - 4.2 Agriculture and agroindustry

Description succincte (Anglais)

See abstract

Partenaires et organisations internationales (Anglais)

Milan University (UNIMI) (I), Uni Brescia Hospital (BRHSP)(I), Schering AG (D)

Résumé des résultats (Abstract) (Anglais)

Overall aim
In this project we intend to develop transgenic reporter animals for the testing of estrogenic compounds. The approach adopted by the UNIFR team was to assemble a multifunctional reporter cassette that comprises an estrogen-inducible and a control readout and to insert this cassette into a gene targeting vector allowing its site-specific integration a constitutively open locus such as the HPRT gene. The homologous integration into appropriate mouse ES cells will generate the prospected reporter transgenic.
Choice of reporter functions
After numerous testing, we have opted for two fusion cassettes. The cassette HA-Zalpha-BLA was assembled by fusing the HA epitope, the LacZ alpha complementing peptide and the ORF encoding beta lactamase (BLA). The second cassette FLAG-Zomega consists of the FLAG epitope fused to the lacZ-omega peptide. Extensive tests assessed that all enzymatic functions and epitope detectability were essentially unimpaired in these constructs. Details are in the scientific report of September 1999 and September 2000.
A quantitative beta lactamase assay
A sensitive and inexpensive quantification method was developed based on the fact that the antibiotic ampicillin is a beta-lactamase substrate. Bacteria were transformed using the engineered plasmid pJOE-tet which confers tetracycline resistance and produces melanin that black-stains transformed E coli.. Transformed bacteria are incubated in LB containing ampicillin and various concentrations of beta-lactamase (from extracts or standards). With this assay the concentration of beta-lactamase conferring bacterial growth can be quantitated by comparison with the standard dilutions. details are under scientific report of September 2000.
Construction of a double reporter combining estrogen-inducible and constitutive readouts
Extensive tests results indicated that the the diERE-SV promoter displayed the highest level of inducibility and this was linked to the HA-Zalpha-BLA function. This inducible construct was linked to the basal control reporter which bears the FLAG-Zomega under constitutive transcription. Thus the final cassette was diERE-HA-Zalpha-BLA-SV linked to CMV-FLAG-Zomega. This could be finally assembled only after the scientific report of september 2000. In this construct the HA immunodetection, the Zalpha complementation and the BLA enzymatic detection can be used (individually or in combination) as estrogen dependent readouts, while the FLAG-LacZ function can be used as internal control readout (by antibody detection either in situ or in vitro).
Final construction and test of the targeting vector
The chosen vector (pMP8SKB) contains murine sequences derived from the region 5' to the HPRT gene, the human HPRT promoter and exon 1 followed by murine sequence encompassing exon 2, 3 and downstream flanking regions. We fully re-sequenced the insert (approx. 11 kilobases) to determine its exact structure and to find the best strategy for inserting our reporter cassette. We then used unique PacI and NotI to engineer the cassette for a total of 22 kb. The replacement vector was demonstrated to maintain estrogen responsiveness without disturbance by the murine flanking sequences (details are in the scientific report January 2001). This vector is the basis for the transformation of E14TG2 ES cells that shall generate the prospected transgenic mouse strains.

Références bases de données (Anglais)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 97.0001