Conditional Inactivation of the Dopamine Receptor 5Gene: Flanking the Drd5 Gene With loxP SitesJoerg Heyer, * Qiurong Xiao, Bozena Bugaj-Gaweda, Sylvie Ramboz, and Axel UnterbeckMemory Pharmaceuticals Corp., Montvale, New Jersey

Received 29 December 2001; Accepted 30 December 2001Published online 13 February 2002

Dopamine is a neurotransmitter that controls many func-tions, among others, locomotor activity and cognition. Ithas been implicated in various neurodegenerative dis-eases such as Parkinson’s disease, schizophrenia, andTourette’s syndrome. Dopamine receptors are G-protein-coupled seven transmembrane proteins. In mammals,five dopamine receptor subtypes are known. The dopa-mine receptor 1-like subfamily (D1-like) consists of theD1 and the D5 receptors, which are pharmacologicallyindistinguishable (Missale et al., 1998). Dopamine recep-tor D1 is more prevalent than the D5 receptor and isexpressed in the brain as well as in various other tissues.In the brain the D5 receptor gene is expressed at muchlower levels than the D1 receptor gene and also shows amore restricted expression profile (Ciliax et al., 2000;Luedtke et al., 1999). Dopamine D5 receptors are foundin the cerebral cortex, thalamus, diagonal band area,striatum, substantia nigra, and hippocampus (Luedtke etal., 1999).

The lack of selective pharmacological tools has pre-cluded isolated evaluation of the role of D1 and D5receptors in central nervous system (CNS) function.Here we present an alternative strategy for approach-ing this problem. The D1-like receptor family of genesare highly homologous and do not contain introns(Vallone et al., 2000). The D5 receptor gene (Drd5)codes for a 475 amino acid protein and is located onchromosome HSA4/MMU5 (Weinshank et al., 1991).Genetic inactivation for the D1-like receptor genes hasbeen reported but no conditional allele (Drago et al.,1994; Sibley, 1999; Xu et al., 1994). Here we presenta conditional Drd5 allele utilizing the phage P1 Cre/loxP system.

We have identified a 10 kbp BamH1 genomic frag-ment that contained the Drd5 gene from a 129/SvJBAC library (Genome Systems, Cambridge, UK).Within this fragment an 8.5 kbp HindIII fragmentrepresents the homology regions of the target vector.We introduced a floxed Hygromycin/Gentamycin se-lection cassette into a NdeI site. The double selectioncassette allows for positive selection in ES-cells (Hy-gromycin) and E. coli (Gentamycin). Additionally thedouble selection cassette can be deleted by the Creenzyme, resulting in a single loxP site in the genomicsequence. If this cassette is used for introducing bothloxP sites, the functionality of the loxP sites is assured.

After inserting the floxed double selection cassetteinto the NdeI site, we passed this subclone throughCre expressing E. coli and deleted the cassette. Thisleft a single loxP site inserted into the NdeI site (Fig.1). The second set of loxP sites, which flanked thedouble selection cassette, was inserted into the EcoRVsite. We left the double resistant cassette in place foruse as the selection marker. The resulting target vec-tor pmuDrd5-tm1Mpc represents the Drd5 geneflanked by loxP sites (Fig. 1). Since Cre-mediated re-combination results in a complete deletion of theDrd5 gene, we expect this to be a null mutation.

The target vector was introduced into 129/Sv/EV MPCES-cells (unpublished data) and clones positive for ho-mologous recombination were selected. Two ES-cellclones were found to be positive and injected into C57/BL6 blastocysts. Both clones gave rise to chimeras andgermline transmission (Fig. 1b).

We functionally tested the loxP sites by deleting theDrd5 gene in Cre expressing E. coli and in ES-cells.Complete deletion of the DRrd5 locus was observed in E.coli (Fig. 2a). We tested in vivo for removal of theselection cassette by transient transfection of Cre intoES-cells carrying the tm1Mpc allele. We found the tm1.1allele, and therefore deletion of the cassette, in 5 out of42 ES-cell colonies (Fig. 2b).

We have generated the loxP site flanked Drd5 alleleDrd5tm1Mpc. The tm1.1Mpc allele allows for condi-tional inactivation of the dopamine receptor 5 gene in atissue- or developmental-specific fashion. We plan toutilize this allele and its derivatives to study the functionof the D5 receptor in cognition and learning.

ACKNOWLEDGMENTS

We thank Daguang Wang, Mike DeVivo, Chris Leonard,and Greg Rose for thoughtful discussions and EleanorSimpson and Eric Kandel for their helpfulness.

* Correspondence to: Joerg Heyer, Memory Pharmaceuticals Corp., 100Philips Parkway, Montvale, NJ 07645.

E-mail: heyer@memorypharma.com

© 2002 Wiley-Liss, Inc. genesis 32:102–104 (2002)DOI 10.1002/gene.10069

FIG. 1. Integration of loxP sites into the Drd5 locus. a: Genomic locus, targeting, and deletion strategy of the Drd5 locus. A 10-kbp genomicBamH1 fragment that contains the Drd5 gene was utilized to construct the target vector pmuDrd5-tm1Mpc. A floxed selection cassette wasintroduced into the NdeI site and consequently removed by the Cre enzyme, leaving a single loxP site inserted into the NdeI site. A secondfloxed selection cassette was introduced into the EcoRV site. pmuDrd5-1Mpc was introduced into ES-cells and the Drd5tm1Mpc allele wasfound in two ES-cell clones. Both were transmitted through the germline. B, BamH1; H, HindIII; E, EcoRV; N, NdeI; black triangles representloxP sites. b: Southern blot analysis of Drd5 alleles in F1 offspring mice. Tail DNA was digested with BamH1, blotted, and hybridized usingthe 5� probe BamH1/HindIII (800 bp fragment). The WT allele is seen as a 3.2 kbp fragment and the tm1Mpc allele is detected as a 6.5 kbpfragment. c: PCR analysis of the Drd5 alleles. Detection of the Drd5 alleles by PCR results in a 210 bp WT fragment (primer J655�-cctttctatgacgtgaattgctg and J67 5�-gaaataggctcaacatccagc) and the tm1Mpc allele results in a 1,577 bp fragment (primer Q1a5�-ctgttctctgtctgaacctc and Q2a 5�-gatcggaattcttgaagacg).

103INACTIVATION OF THE DOPAMINE RECEPTOR 5 GENE

LITERATURE CITED

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Drago J, Gerfen CR, Lachowicz JE, Steiner H, Hollon TR, Love PE, OoiGT, Grinberg A, Lee EJ, Huang SP, et al. 1994. Altered striatalfunction in a mutant mouse lacking D1A dopamine receptors.Proc Natl Acad Sci USA 91:12564–12568.

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Xu M, Moratalla R, Gold LH, Hiroi N, Koob GF, Graybiel AM, TonegawaS. 1994. Dopamine D1 receptor mutant mice are deficient instriatal expression of dynorphin and in dopamine-mediated behav-ioral responses. Cell 79:729–742.

FIG. 2. Deletion of the Drd5 gene. a: Cre-expressing bacteria weretransformed with the vector pmuDrd5-tm1Mpc. Plasmids were iso-lated from the surviving colonies and analyzed by restriction diges-tion and PCR (data not shown). The target vector, representing thetm1Mpc allele, gives rise to diagnostic 8 kbp XhoI and 11kbp HindIIIbands. The deleted plasmid, representing the tm1.2Mpc allele,gives rise to a diagnostic 7 kbp XhoI and a 3.8 kbp HindIII fragment.X, XhoI; H, HindIII; M, marker; black triangles represent loxP sites; �,deleted allele. b: BamH1 Southern analysis of ES-cell coloniestransiently transfected with Cre. Clone 25 harbors a WT and atm1Mpc allele, whereas clone 76 harbors a WT and a tm1.1Mpcallele. The allele sizes are: WT 3.2 kbp, tm1Mpc 6.5 kbp, tm1.1Mpc3.5 kbp.

104 HEYER ET AL.