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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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Labeling of subsets of neurons in different hsp68-nlacZ lines.(A) Coronal section of a mouse carrying a single copy of the hsp68-nlacZ transgene that integrated into the coding region of the Npas3 gene (bar = 1 mm). (B) The table shows the genomic insertion site of eight mouse lines carrying a single copy of the hsp68-nlacZ transgene. (C) Sagittal sections of a mouse carrying a single copy of the hsp68-nlacZ transgene that integrated into the coding region of the Phospholipase C-gamma1 gene. Medial to lateral sections of the brain reveal a medio-lateral expression gradient both in the hippocampus and cerebellum (bar = 1mm).
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pone-0038593-g003: Labeling of subsets of neurons in different hsp68-nlacZ lines.(A) Coronal section of a mouse carrying a single copy of the hsp68-nlacZ transgene that integrated into the coding region of the Npas3 gene (bar = 1 mm). (B) The table shows the genomic insertion site of eight mouse lines carrying a single copy of the hsp68-nlacZ transgene. (C) Sagittal sections of a mouse carrying a single copy of the hsp68-nlacZ transgene that integrated into the coding region of the Phospholipase C-gamma1 gene. Medial to lateral sections of the brain reveal a medio-lateral expression gradient both in the hippocampus and cerebellum (bar = 1mm).

Mentions: In order to test the applicability of enhancer detection by lentiviral vectors, we first generated a lentiviral enhancer probe containing the minimal promoter of the human heat-shock gene 68 (hsp68), which by itself has a very low basal level of activity and must be activated by an enhancer on the same chromosome to achieve expression of a reporter gene [12]. We then generated transgenic mice by infecting single-cell mouse embryos with a recombinant lentiviral vector carrying the hsp68 minimal promoter controlling the expression of the nlacZ gene, which contains a nuclear localization sequence. Treated embryos were then returned to the uterus of surrogate mothers to complete development. Transgenic founders were bred with wild type animals to obtain a single copy of an insertion in the offspring. To maximize the chances of obtaining animals in which single-copy transgenes could be generated within one breeding cycle, we titrated the lentiviral vector in our procedure so that we obtained 1–4 insertions per founder animal that could be separated by breeding over 1–2 generations. Most animals (23 out of 25 lines) carrying a single copy insertion displayed distinct expression patterns in the brain when assayed by lacZ histochemistry. The pattern of nlacZ-expressing cells in the different transgenic lines ranged from near-ubiquitous to restricted to, for example, specific layers in the cortex, subregions of the hippocampus, and medio-lateral gradients within the same structure (Figure 2). We cloned the insertion site of eight different lines by ligation-mediated PCR. As expected from previously published works [8], four out eight insertions were located within introns, and the remaining four insertions were located either upstream or downstream of the genes’ coding region (Figure 3).


Genetic labeling of neuronal subsets through enhancer trapping in mice.

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

Labeling of subsets of neurons in different hsp68-nlacZ lines.(A) Coronal section of a mouse carrying a single copy of the hsp68-nlacZ transgene that integrated into the coding region of the Npas3 gene (bar = 1 mm). (B) The table shows the genomic insertion site of eight mouse lines carrying a single copy of the hsp68-nlacZ transgene. (C) Sagittal sections of a mouse carrying a single copy of the hsp68-nlacZ transgene that integrated into the coding region of the Phospholipase C-gamma1 gene. Medial to lateral sections of the brain reveal a medio-lateral expression gradient both in the hippocampus and cerebellum (bar = 1mm).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369840&req=5

pone-0038593-g003: Labeling of subsets of neurons in different hsp68-nlacZ lines.(A) Coronal section of a mouse carrying a single copy of the hsp68-nlacZ transgene that integrated into the coding region of the Npas3 gene (bar = 1 mm). (B) The table shows the genomic insertion site of eight mouse lines carrying a single copy of the hsp68-nlacZ transgene. (C) Sagittal sections of a mouse carrying a single copy of the hsp68-nlacZ transgene that integrated into the coding region of the Phospholipase C-gamma1 gene. Medial to lateral sections of the brain reveal a medio-lateral expression gradient both in the hippocampus and cerebellum (bar = 1mm).
Mentions: In order to test the applicability of enhancer detection by lentiviral vectors, we first generated a lentiviral enhancer probe containing the minimal promoter of the human heat-shock gene 68 (hsp68), which by itself has a very low basal level of activity and must be activated by an enhancer on the same chromosome to achieve expression of a reporter gene [12]. We then generated transgenic mice by infecting single-cell mouse embryos with a recombinant lentiviral vector carrying the hsp68 minimal promoter controlling the expression of the nlacZ gene, which contains a nuclear localization sequence. Treated embryos were then returned to the uterus of surrogate mothers to complete development. Transgenic founders were bred with wild type animals to obtain a single copy of an insertion in the offspring. To maximize the chances of obtaining animals in which single-copy transgenes could be generated within one breeding cycle, we titrated the lentiviral vector in our procedure so that we obtained 1–4 insertions per founder animal that could be separated by breeding over 1–2 generations. Most animals (23 out of 25 lines) carrying a single copy insertion displayed distinct expression patterns in the brain when assayed by lacZ histochemistry. The pattern of nlacZ-expressing cells in the different transgenic lines ranged from near-ubiquitous to restricted to, for example, specific layers in the cortex, subregions of the hippocampus, and medio-lateral gradients within the same structure (Figure 2). We cloned the insertion site of eight different lines by ligation-mediated PCR. As expected from previously published works [8], four out eight insertions were located within introns, and the remaining four insertions were located either upstream or downstream of the genes’ coding region (Figure 3).

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