The identification of cis-sequences responsible for spatiotemporal patterns of gene expression often requires the functional analysis of large genomic regions. In this study a 100-kb zebrafish Hoxa-11b-lacZ reporter gene was constructed and expressed in transgenic mice. PAC clone 10-O19, containing a portion of the zebrafish HoxA-b cluster, was captured into the yeast-bacterial shuttle vector, pPAC-ResQ, by recombinogenic targeting. A lacZ reporter gene was then inserted in-frame into exon 1 of the zfHoxa-11b locus by a second round of recombinogenic targeting. Expression of the zfHoxa-11b-lacZ reporter gene in 10.5 d.p.f. transgenic mouse embryos was observed only in the posterior portion of the A-P axis, in the paraxial mesoderm, neural tube, and somites. These findings demonstrate the utility of recombinogenic targeting for the modification and expression of large inserts captured from P1/PAC clones.
Bibliographical noteFunding Information:
Acknowledgements We thank Caroline B. Long and Elizabeth Mongillo for technical assistance. C.H.C. is the recipient of an Alfred P. Sloan/National Science Foundation Postdoctoral Research Fellowship in Molecular Evolution. Financial support through National Science Foundation grants IBN-9630567 to G.P.W., F.H.R., C.T.A.; IBN-9614940 to C.T.A.; as well as a National Institutes of Health grant AI 39008-01 to C.T.A. is gratefully appreciated.
- AbDb-like Hox genes
- Cis-regulatory sequences
- Homologous recombination
- P1/PAC clones
- Reporter gene