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

Chi Hua Chiu; Chris T. Amemiya; Janet L. Carr; Jaya Bhargava; Joseph K. Hwang; Cooduvalli S. Shashikant; Frank H. Ruddle; Günter P. Wagner

doi:10.1007/s004270050016

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

Chi Hua Chiu, Chris T. Amemiya, Janet L. Carr, Jaya Bhargava, Joseph K. Hwang, Cooduvalli S. Shashikant, Frank H. Ruddle, Günter P. Wagner

Obstetrics, Gynecology and Women's Health - Fetal Medicine

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15 Scopus citations

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.

Original language	English (US)
Pages (from-to)	105-109
Number of pages	5
Journal	Development Genes and Evolution
Volume	210
Issue number	2
DOIs	https://doi.org/10.1007/s004270050016
State	Published - 2000

Bibliographical note

Funding 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.

Keywords

AbDb-like Hox genes
Cis-regulatory sequences
Homologous recombination
P1/PAC clones
Reporter gene

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10.1007/s004270050016

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Chiu, C. H., Amemiya, C. T., Carr, J. L., Bhargava, J., Hwang, J. K., Shashikant, C. S., Ruddle, F. H., & Wagner, G. P. (2000). 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. Development Genes and Evolution, 210(2), 105-109. https://doi.org/10.1007/s004270050016

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. / Chiu, Chi Hua; Amemiya, Chris T.; Carr, Janet L.; Bhargava, Jaya; Hwang, Joseph K.; Shashikant, Cooduvalli S.; Ruddle, Frank H.; Wagner, Günter P.

In: Development Genes and Evolution, Vol. 210, No. 2, 2000, p. 105-109.

Research output: Contribution to journal › Article › peer-review

Chiu, CH, Amemiya, CT, Carr, JL, Bhargava, J, Hwang, JK, Shashikant, CS, Ruddle, FH & Wagner, GP 2000, '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', Development Genes and Evolution, vol. 210, no. 2, pp. 105-109. https://doi.org/10.1007/s004270050016

Chiu, Chi Hua ; Amemiya, Chris T. ; Carr, Janet L. ; Bhargava, Jaya ; Hwang, Joseph K. ; Shashikant, Cooduvalli S. ; Ruddle, Frank H. ; Wagner, Günter P. / 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. In: Development Genes and Evolution. 2000 ; Vol. 210, No. 2. pp. 105-109.

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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.",

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note = "Funding 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.",

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AU - Amemiya, Chris T.

AU - Carr, Janet L.

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AU - Shashikant, Cooduvalli S.

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N2 - 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.

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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

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