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.
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.
Chiu
C H
CH
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
Amemiya
C T
CT
Carr
J L
JL
Bhargava
J
J
Hwang
J K
JK
Shashikant
C S
CS
Ruddle
F H
FH
Wagner
G P
GP
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
D004274:DNA, Recombinant; D018398:Homeodomain Proteins; C094041:Hoxa11 protein, mouse; D029961:Zebrafish Proteins; C491346:hoxa11b protein, zebrafish