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 |
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| Authors: | C-H Chiu C T Amemiya J L Carr J Bhargava J K Hwang C S Shashikant F H Ruddle G P Wagner |
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| Institution: | (1) Department of Ecology and Evolutionary Biology, Osborn Memorial Laboratory 327, Yale University, P.O. Box 208106, New Haven, CT 06520-8106, USA e-mail: chi-hua.chiu@yale.edu Tel.: +1-203-4329999, Fax: +1-203-4323870, US;(2) Center for Human Genetics, Boston University School of Medicine, 700 Albany Street, W408, Boston, MA 02118, USA, US;(3) Genaissance Pharmaceuticals Inc., 5 Science Park, New Haven, CT 06511, USA, US;(4) Department of Obstetrics and Gynecology, Yale University, School of Medicine, P.O. Box 208063, New Haven, CT 06520, USA, US;(5) Department of Dairy and Animal Science, College of Agricultural Sciences, Pennsylvania State University, 324 Henning Building, University Park, PA 16802-3503, USA, US;(6) Departments of Molecular, Cellular, and Developmental Biology, Kline Biology Tower, Yale University, P.O. Box 208103, New Haven, CT 06520, USA, US;(7) Departments of Molecular, Cellular, and Developmental Biology and of Genetics, Kline Biology Tower, Yale University, P.O. Box 208103, New Haven, CT 06520, USA, US |
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| 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.
Received: 22 June 1999 / Accepted: 1 September 1999 |
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| Keywords: | Cis-regulatory sequences P1/PAC clones Homologous recombination Reporter gene AbDb-like Hox genes |
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