Article

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

In Vitro Cellular & Developmental Biology - Plant (impact factor: 1.5). 04/2012; 37(1):1-5. DOI:10.1007/s11627-001-0001-6 pp.1-5

ABSTRACT Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied
in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine
and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding
endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized
probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and
multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol
was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.

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    Article: In situ methods to localize transgenes and transcripts in interphase nuclei: a tool for transgenic plant research
    Ana Santos, Eva Wegel, George Allen, William Thompson, Eva Stoger, Peter Shaw, Rita Abranches
    [show abstract] [hide abstract]
    ABSTRACT: Abstract Genetic engineering of commercially important crops has become routine in many laboratories. However, the inability to predict where a transgene will integrate and to efficiently select plants with stable levels of transgenic expression remains a limitation of this technology. Fluorescence in situ hybridization (FISH) is a powerful technique that can be used to visualize transgene integration sites and provide a better understanding of transgene behavior. Studies using FISH to characterize transgene integration have focused primarily on metaphase chromosomes, because the number and position of integration sites on the chromosomes are more easily determined at this stage. However gene (and transgene) expression occurs mainly during interphase. In order to accurately predict the activity of a transgene, it is critical to understand its location and dynamics in the three-dimensional interphase nucleus. We and others have developed in situ methods to visualize transgenes (including single copy genes) and their transcripts during interphase from different tissues and plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis, and extend characterization to the interphase nucleus, thus increasing the likelihood of accurate prediction of transgene activity. Furthermore, this approach is useful for studying nuclear organization and the dynamics of genes and chromatin.
    Plant Methods. 01/2006;
  • Source
    Article: In situ methods to localize transgenes and transcripts in interphase nuclei: a tool for transgenic plant research.
    Ana Paula Santos, Eva Wegel, George C Allen, William F Thompson, Eva Stoger, Peter Shaw, Rita Abranches
    [show abstract] [hide abstract]
    ABSTRACT: Genetic engineering of commercially important crops has become routine in many laboratories. However, the inability to predict where a transgene will integrate and to efficiently select plants with stable levels of transgenic expression remains a limitation of this technology. Fluorescence in situ hybridization (FISH) is a powerful technique that can be used to visualize transgene integration sites and provide a better understanding of transgene behavior. Studies using FISH to characterize transgene integration have focused primarily on metaphase chromosomes, because the number and position of integration sites on the chromosomes are more easily determined at this stage. However gene (and transgene) expression occurs mainly during interphase. In order to accurately predict the activity of a transgene, it is critical to understand its location and dynamics in the three-dimensional interphase nucleus. We and others have developed in situ methods to visualize transgenes (including single copy genes) and their transcripts during interphase from different tissues and plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis, and extend characterization to the interphase nucleus, thus increasing the likelihood of accurate prediction of transgene activity. Furthermore, this approach is useful for studying nuclear organization and the dynamics of genes and chromatin.
    Plant Methods 02/2006; 2:18. · 2.83 Impact Factor
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Keywords

chromosomes
 
cy3-conjugated anti-mouse IgG
 
denatured rice metaphase chromosomes
 
digoxygenin-labeled endogenous
 
endogenous sequences
 
FISH protocol
 
Fluorescence
 
fluorescence microscopy
 
genes
 
integration loci
 
karyotyping
 
localizing single copy genes
 
meristem tissue
 
Oryza sativa L
 
powerful tool
 
sensitive FISH procedure
 
sequences
 
Single copy
 
situ hybridization
 
T-DNA sequences