Cre Recombinase Expression in the Floorplate, Notochord and Gut Epithelium in Transgenic Embryos Driven by the Hoxa-1 Enhancer III Xue Li and Thomas Lufkin* Brookdale Center for Developmental and Molecular Biology, Mount Sinai School of Medicine, New York, New York Received October 1999; Accepted 5 October 1999 The Hoxa-1 homeobox gene functions as a director of CNS patterning beginning at the neural plate stage of embryogenesis, and in the absence of Hoxa-1 activity, several hindbrain rhombomeres fail to undergo normal development (Carpenter et al., 1993; Lufkin et al., 1991; Mark et al., 1993). The Hoxa-1 gene has three well- characterized enhancers (I-III), which together are nec- essary for recapitulating the full expression pattern of Hoxa-1 in transgenic embryos (Frasch et al., 1995). Individually each of the enhancers gives a defined subset of the Hoxa-1 expression domain. The 0.5 kb Hoxa-1 Enhancer III is located 3' to the Hoxa-1 polyadenylation site and contains a functional retinoic acid regulatory element (RARE), which is necessary for its activity and for driving expression of the Hoxa-1 locus in neuroepi- thelium (Frasch et al., 1995, Fig 1.). The activity of Enhancer III has been examined in over 14 expressing stable transgenic lines and 12 expressing transient F0 embryos. Ten of the 14 stable lines and 10 of the 12 transient F0 embryos gave an identical pattern of trans- gene expression (Frasch et al., 1995; Xue Li, 1998) showing activity in the floorplate, notochord, and gut epithelium beginning at E8.5 and continuing strongly through E12.5, followed by a decrease to background levels by E14.5 (Fig. 2). The Enhancer III-Cre (EIII-Cre) construct (Fig. 1) has the Hoxa-1 Enhancer III driving transcription from a basal TATA box promoter (Frasch et al., 1995). The dicistronic mRNA has the Cre recombinase ORF (Sauer, 1987) at the 5' end, followed by the human placental alkaline phosphatase (AP) ORF (Cepko et al., 1993), which is linked to an IRES sequence to allow cap-independent translation initiation as previously described (Kim et al., 1992; Li et al., 1997). The sequence surrounding the ATG start codon of the Cre ORF was modified by site-directed mutagenesis (Deng and Nick- oloff, 1992) to more closely resemble a consensus Kozak sequence (Kozak, 1991). The transgene mRNA is pro- cessed at the 3' end by an SV40 polyadenylation signal (Frasch et al., 1995). Five EnIII-Cre transgenic founder lines were generated. Four of the EnIII-Cre transgenics lines (Lines 1, 4 – 6) gave the predicted restriction fragment banding pattern when digested with BamHI and probed with the Cre and AP ORF fragments (Fig. 2). Lines 1, 4, and 5 expressed Cre and AP, and lines 1 and 5 displayed an identical pattern of Cre and AP expression, which was characteristic for the Hoxa-1 Enhancer III (Fig. 1). ACKNOWLEDGMENTS We would like to thank Heidi Stuhlmann for providing the Alkaline Phosphatase cDNA, Brian Sauer for the Cre recombinase cDNA, and Hee-Sup Shin for the IRES se- quence. LITERATURE CITED Carpenter EM, Goddard JM, Chisaka O, Manley NR, Capecchi MR. 1993. Loss of Hox-A1 (Hox-1.6) function results in the reorgani- zation of the murine hindbrain. Development 118:1063–1075. Cepko CL, Ryder EF, Austin CP, Walsh C, Fekete DM. 1993. Lineage analysis using retrovirus vectors. Meth Enzymol 225:933–960. Deng WP, Nickoloff JA. 1992. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem 200:81– 88. Frasch M, Chen X, Lufkin T. 1995. Evolutionary-conserved enhancers direct region-specific expression of the murine Hoxa-1 and Hoxa-2 loci in both mice and Drosophila. Development 121:957–974. Kim DG, Kang HM, Jang SK, Shin HS. 1992. Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus. Mol Cell Biol 12:3636 –3643. Kozak M. 1991. An analysis of vertebrate mRNA sequences: Intimations of translational control. J Cell Biol 115:887–903. Li X, Wang W, Lufkin T. 1997. Dicistronic lacZ and alkaline phosphatase reporter constructs permit simultaneous histological analysis of ex- pression from multiple transgenes. BioTechniques 23:874 – 882. Li X. 1998. A Cre/loxP binary genetic approach to study murine spinal cord and limb development. PhD Thesis, Mt. Sinai School of Medicine, New York, NY. Lufkin T, Dierich A, LeMeur M, Mark M, Chambon P. 1991. Disruption of the Hox-1.6 homeobox gene results in defects in a region corre- sponding to its rostral domain of expression. Cell 66:1105–1119. Lufkin T, Mark M, Hart CP, LeMeur M, Chambon P. 1992. Homeotic transformation of the occipital bones of the skull by ectopic expression of a homeobox gene. Nature 359:835– 841. Mark M, Lufkin T, Vonesch J, Ruberte E, Olivo J, Dolle ´ P, Gorry P, Lumsden A, Chambon P. 1993. Two rhombomeres are altered in Hoxa-1 mutant mice. Development 119:319 –338. Sauer B. 1987. Functional expression of the cre-lox site-specific recom- bination system in the yeast Saccharomyces cerevisiae. Mol Cell Biol 7:2087–2096. * Correspondence to: Thomas Lufkin, Brookdale Center for Developmen- tal and Molecular Biology, Mount Sinai School of Medicine, Box 1020, One Gustave L. Levy Place, New York, NY 10029-6574. E-mail: lufkit01@doc.mssm.edu © 2000 Wiley-Liss, Inc. genesis 26:121–122 (2000)