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Genetic labeling of neuronal subsets through enhancer trapping in mice.

Kelsch W, Stolfi A, Lois C - PLoS ONE (2012)

Bottom Line: The ability to label, visualize, and manipulate subsets of neurons is critical for elucidating the structure and function of individual cell types in the brain.We have developed an enhancer trap strategy in mammals by generating transgenic mice with lentiviral vectors carrying single-copy enhancer-detector probes encoding either the marker gene lacZ or Cre recombinase.This transgenic strategy allowed us to genetically identify a wide variety of neuronal subpopulations in distinct brain regions.

View Article: PubMed Central - PubMed

Affiliation: Bernstein Center for Computational Neuroscience, CIMH, Medical Faculty Mannheim, University Heidelberg, Heidelberg, Germany.

ABSTRACT
The ability to label, visualize, and manipulate subsets of neurons is critical for elucidating the structure and function of individual cell types in the brain. Enhancer trapping has proved extremely useful for the genetic manipulation of selective cell types in Drosophila. We have developed an enhancer trap strategy in mammals by generating transgenic mice with lentiviral vectors carrying single-copy enhancer-detector probes encoding either the marker gene lacZ or Cre recombinase. This transgenic strategy allowed us to genetically identify a wide variety of neuronal subpopulations in distinct brain regions. Enhancer detection by lentiviral transgenesis could thus provide a complementary method for generating transgenic mouse libraries for the genetic labeling and manipulation of neuronal subsets.

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Related in: MedlinePlus

Generation of transgenic mice carrying a nlacZ enhancer probe under control of a hsp68 minimal promoter.Enhancer probes integrate into different sites in the genome. Depending on the site of integration the interaction of the introduced minimal promoter and enhancer elements of the genome results in restricted expression of the transgene.
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pone-0038593-g001: Generation of transgenic mice carrying a nlacZ enhancer probe under control of a hsp68 minimal promoter.Enhancer probes integrate into different sites in the genome. Depending on the site of integration the interaction of the introduced minimal promoter and enhancer elements of the genome results in restricted expression of the transgene.

Mentions: Mammalian brains contain a bewildering variety of different classes of neurons. Interestingly, it is still not known how many different neuronal types exist in the mouse or rat brain, the most commonly used laboratory animals. The number of distinct neuronal types steadily increases as new analyses and techniques better differentiate those using combined anatomical, electrophysiological, and genetic criteria [1], [2], [3]. The ability to identify specific neuronal types will be critical for understanding their contribution towards brain function and behavior [4]. Moreover, to investigate the function of genetically defined subsets of cells, it is necessary not only to visualize them, but also to selectively manipulate their gene expression. Transgenic animals expressing fluorescent markers in neuronal subsets [5] have proven very useful for in vivo imaging and electrophysiology. However, there is currently only a limited number of mouse lines that express visible markers in subsets of neurons [5], [6], and only a few lines can be used for selective gene manipulation in these neuronal populations [7]. We have developed a new mouse transgenic strategy in which expression of the recombinase Cre depends on enhancer detection, with the goal of creating libraries of transgenic mice with the ability to visualize and manipulate genes in selective subsets of neurons. To achieve this goal, we used lentiviral transgenesis to deliver enhancer-detection probes into single-cell mouse embryos. Lentiviruses integrate preferentially into gene-rich regions of the genome [8], thereby increasing the chance that a transgene insertion will be activated by nearby enhancers. The strategy of enhancer detection relies on a gene of choice present within a transgenic probe whose transcription depends on where the probe integrates in the genome. In eukaryotic cells, gene expression depends on the presence of cis-DNA sequences that regulate the rate of transcription of the gene and transcription factors that recognize them [9]. The two major classes of DNA cis-regulatory transcriptional elements are long-range and short-range elements. Short-range regulatory elements, called promoters, are located immediately upstream of the gene they regulate. In contrast, long-range regulatory elements can be located either within introns, upstream or downstream of the transcription start site, sometimes up to hundreds of kilobases away from the gene whose activity they regulate [10]. These distant regulatory elements can either have a positive or a negative effect on transcription, and they are designated as enhancers or silencers, respectively. In line with the traditional nomenclature used in enhancer trapping, here we will refer to both of these elements as enhancers for simplicity. By themselves, neither enhancers nor promoters are sufficient to drive transcription. The functional expression of genes requires the combined activity of enhancers and promoters, situated in a specific configuration with respect to the gene they regulate. The strategy of enhancer detection is based on the requirement of promoters to be activated by enhancers to achieve expression of the genes they regulate [11] (Figure 1). We have engineered lentiviral vectors encoding enhancer detection probes that allowed efficient generation of transgenic mice selectively expressing Cre under the influence of enhancers located in the vicinity of the chromosomal integration site. We demonstrated the utility of these transgenic mice for the identification of specific neuronal classes and for their electrophysiological characterization. In addition, these mice can be used to perform Cre-mediated gene manipulation in selective neuronal types. Our results demonstrate that enhancer detection, a technique that has proven extremely valuable to genetically identify and manipulate cells in invertebrates, could provide a complementary approach to genetically dissection of the neuronal diversity of the mouse brain.


Genetic labeling of neuronal subsets through enhancer trapping in mice.

Kelsch W, Stolfi A, Lois C - PLoS ONE (2012)

Generation of transgenic mice carrying a nlacZ enhancer probe under control of a hsp68 minimal promoter.Enhancer probes integrate into different sites in the genome. Depending on the site of integration the interaction of the introduced minimal promoter and enhancer elements of the genome results in restricted expression of the transgene.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369840&req=5

pone-0038593-g001: Generation of transgenic mice carrying a nlacZ enhancer probe under control of a hsp68 minimal promoter.Enhancer probes integrate into different sites in the genome. Depending on the site of integration the interaction of the introduced minimal promoter and enhancer elements of the genome results in restricted expression of the transgene.
Mentions: Mammalian brains contain a bewildering variety of different classes of neurons. Interestingly, it is still not known how many different neuronal types exist in the mouse or rat brain, the most commonly used laboratory animals. The number of distinct neuronal types steadily increases as new analyses and techniques better differentiate those using combined anatomical, electrophysiological, and genetic criteria [1], [2], [3]. The ability to identify specific neuronal types will be critical for understanding their contribution towards brain function and behavior [4]. Moreover, to investigate the function of genetically defined subsets of cells, it is necessary not only to visualize them, but also to selectively manipulate their gene expression. Transgenic animals expressing fluorescent markers in neuronal subsets [5] have proven very useful for in vivo imaging and electrophysiology. However, there is currently only a limited number of mouse lines that express visible markers in subsets of neurons [5], [6], and only a few lines can be used for selective gene manipulation in these neuronal populations [7]. We have developed a new mouse transgenic strategy in which expression of the recombinase Cre depends on enhancer detection, with the goal of creating libraries of transgenic mice with the ability to visualize and manipulate genes in selective subsets of neurons. To achieve this goal, we used lentiviral transgenesis to deliver enhancer-detection probes into single-cell mouse embryos. Lentiviruses integrate preferentially into gene-rich regions of the genome [8], thereby increasing the chance that a transgene insertion will be activated by nearby enhancers. The strategy of enhancer detection relies on a gene of choice present within a transgenic probe whose transcription depends on where the probe integrates in the genome. In eukaryotic cells, gene expression depends on the presence of cis-DNA sequences that regulate the rate of transcription of the gene and transcription factors that recognize them [9]. The two major classes of DNA cis-regulatory transcriptional elements are long-range and short-range elements. Short-range regulatory elements, called promoters, are located immediately upstream of the gene they regulate. In contrast, long-range regulatory elements can be located either within introns, upstream or downstream of the transcription start site, sometimes up to hundreds of kilobases away from the gene whose activity they regulate [10]. These distant regulatory elements can either have a positive or a negative effect on transcription, and they are designated as enhancers or silencers, respectively. In line with the traditional nomenclature used in enhancer trapping, here we will refer to both of these elements as enhancers for simplicity. By themselves, neither enhancers nor promoters are sufficient to drive transcription. The functional expression of genes requires the combined activity of enhancers and promoters, situated in a specific configuration with respect to the gene they regulate. The strategy of enhancer detection is based on the requirement of promoters to be activated by enhancers to achieve expression of the genes they regulate [11] (Figure 1). We have engineered lentiviral vectors encoding enhancer detection probes that allowed efficient generation of transgenic mice selectively expressing Cre under the influence of enhancers located in the vicinity of the chromosomal integration site. We demonstrated the utility of these transgenic mice for the identification of specific neuronal classes and for their electrophysiological characterization. In addition, these mice can be used to perform Cre-mediated gene manipulation in selective neuronal types. Our results demonstrate that enhancer detection, a technique that has proven extremely valuable to genetically identify and manipulate cells in invertebrates, could provide a complementary approach to genetically dissection of the neuronal diversity of the mouse brain.

Bottom Line: The ability to label, visualize, and manipulate subsets of neurons is critical for elucidating the structure and function of individual cell types in the brain.We have developed an enhancer trap strategy in mammals by generating transgenic mice with lentiviral vectors carrying single-copy enhancer-detector probes encoding either the marker gene lacZ or Cre recombinase.This transgenic strategy allowed us to genetically identify a wide variety of neuronal subpopulations in distinct brain regions.

View Article: PubMed Central - PubMed

Affiliation: Bernstein Center for Computational Neuroscience, CIMH, Medical Faculty Mannheim, University Heidelberg, Heidelberg, Germany.

ABSTRACT
The ability to label, visualize, and manipulate subsets of neurons is critical for elucidating the structure and function of individual cell types in the brain. Enhancer trapping has proved extremely useful for the genetic manipulation of selective cell types in Drosophila. We have developed an enhancer trap strategy in mammals by generating transgenic mice with lentiviral vectors carrying single-copy enhancer-detector probes encoding either the marker gene lacZ or Cre recombinase. This transgenic strategy allowed us to genetically identify a wide variety of neuronal subpopulations in distinct brain regions. Enhancer detection by lentiviral transgenesis could thus provide a complementary method for generating transgenic mouse libraries for the genetic labeling and manipulation of neuronal subsets.

Show MeSH
Related in: MedlinePlus