title: A recombinogenic targeting method to modify large-inserts for cis-regulatory analysis in transgenic mice: construction and expression of a 100-kb, zebrafish Hoxa-11b-lacZ reporter gene.
abstract: 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.
authors.affiliation: Department of Ecology and Evolutionary Biology, Osborn Memorial Laboratory 327, Yale University, P.O. Box 208106, New Haven, CT 06520-8106, USA. chi-hua.chiu@yale.edu
authors.forename: C H
authors.initials: CH
authors.lastname: Chiu
authors.forename: C T
authors.initials: CT
authors.lastname: Amemiya
authors.forename: J L
authors.initials: JL
authors.lastname: Carr
authors.forename: J
authors.initials: J
authors.lastname: Bhargava
authors.forename: J K
authors.initials: JK
authors.lastname: Hwang
authors.forename: C S
authors.initials: CS
authors.lastname: Shashikant
authors.forename: F H
authors.initials: FH
authors.lastname: Ruddle
authors.forename: G P
authors.initials: GP
authors.lastname: Wagner
mesh_terms: D000818:Animals; D004274:DNA, Recombinant; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics*; D007763:Lac Operon; D051379:Mice; D008822:Mice, Transgenic; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins
chemical_list: D004274:DNA, Recombinant; D018398:Homeodomain Proteins; C094041:Hoxa11 protein, mouse; D029961:Zebrafish Proteins; C491346:hoxa11b protein, zebrafish