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The relative contribution of proximal 5' flanking sequence and microsatellite variation on brain vasopressin 1a receptor (Avpr1a) gene expression and behavior.

Donaldson ZR, Young LJ - PLoS Genet. (2013)

Bottom Line: Previous work has suggested that both the proximal 5' flanking region and a polymorphic microsatellite element within that region of the vole Avpr1a gene are associated with variation in V1a receptor (V1aR) distribution and behavior, but neither has been causally linked.This is the first direct evidence that polymorphic microsatellite elements near behaviorally relevant genes can contribute to diversity in brain gene expression profiles, providing a mechanism for generating behavioral diversity both at the individual and species level.However, our results suggest that many features of species-specific expression patterns are mediated by elements outside of the immediate 5' flanking region of the gene.

View Article: PubMed Central - PubMed

Affiliation: Division of Integrative Neuroscience, Department of Psychiatry, Columbia University, New York, New York, United States of America. zoe.donaldson@gmail.com

ABSTRACT
Certain genes exhibit notable diversity in their expression patterns both within and between species. One such gene is the vasopressin receptor 1a gene (Avpr1a), which exhibits striking differences in neural expression patterns that are responsible for mediating differences in vasopressin-mediated social behaviors. The genomic mechanisms that contribute to these remarkable differences in expression are not well understood. Previous work has suggested that both the proximal 5' flanking region and a polymorphic microsatellite element within that region of the vole Avpr1a gene are associated with variation in V1a receptor (V1aR) distribution and behavior, but neither has been causally linked. Using homologous recombination in mice, we reveal the modest contribution of proximal 5' flanking sequences to species differences in V1aR distribution, and confirm that variation in V1aR distribution impacts stress-coping in the forced swim test. We also demonstrate that the vole Avpr1a microsatellite structure contributes to Avpr1a expression in the amygdala, thalamus, and hippocampus, mirroring a subset of the inter- and intra-species differences observed in central V1aR patterns in voles. This is the first direct evidence that polymorphic microsatellite elements near behaviorally relevant genes can contribute to diversity in brain gene expression profiles, providing a mechanism for generating behavioral diversity both at the individual and species level. However, our results suggest that many features of species-specific expression patterns are mediated by elements outside of the immediate 5' flanking region of the gene.

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Targeting vector design.(A) shows the targeting vector used to replace the 5′ flanking region of the mouse Avpr1a gene with corresponding sequences from the prairie vole. We generated three targeting vectors that were identical except for the microsatellite region they contained, which is indicated by the cross hatched region. Triangles denote loxP sites. (B) shows hybridization of the external Southern probe in correctly targeted recombinants for all three lines. Because the Acc651 site used to screen for recombinant stem cells was located within the floxed region, excision of the NeoR also resulted in the recombinant allele yielding a ∼9.5 kb band when detected with the external probe. (C) shows PCR genotyping. All three lines were backcrossed to a C57Bl/6J background for at least 5 generations prior to neuroanatomical and behavioral experiments.
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pgen-1003729-g002: Targeting vector design.(A) shows the targeting vector used to replace the 5′ flanking region of the mouse Avpr1a gene with corresponding sequences from the prairie vole. We generated three targeting vectors that were identical except for the microsatellite region they contained, which is indicated by the cross hatched region. Triangles denote loxP sites. (B) shows hybridization of the external Southern probe in correctly targeted recombinants for all three lines. Because the Acc651 site used to screen for recombinant stem cells was located within the floxed region, excision of the NeoR also resulted in the recombinant allele yielding a ∼9.5 kb band when detected with the external probe. (C) shows PCR genotyping. All three lines were backcrossed to a C57Bl/6J background for at least 5 generations prior to neuroanatomical and behavioral experiments.

Mentions: The targeting strategy is illustrated in Figure 2. For the meadow line, we screened 288 ES cell clones and identified 1 recombinant. For the prairie short line, 192 clones yielded 2 correct recombinants, and for the prairie long line, 288 clones yielded 2 correct recombinants. This corresponds with an overall recombination efficiency of 0.6% (5 of 768). The floxed PGK-NeoR cassette was successfully removed via breeding to a ubiquitously expressing EIIa-Cre recombinase line as confirmed by PCR and Southern Blot (Figure 2). Because the Acc651 site used to screen for recombinant stem cells was located within the floxed region, excision of the NeoR also resulted in the recombinant allele yielding a ∼9.5 kb band when detected with the external probe. The three resulting recombinant alleles, prairie vole long (pvKI-long), prairie vole short (pvKI-short), or meadow vole (mvKI) were identical in sequence except for the composition of the microsatellite element. All three lines were backcrossed to a C57Bl/6J background for at least 5 generations prior to neuroanatomical and behavioral experiments.


The relative contribution of proximal 5' flanking sequence and microsatellite variation on brain vasopressin 1a receptor (Avpr1a) gene expression and behavior.

Donaldson ZR, Young LJ - PLoS Genet. (2013)

Targeting vector design.(A) shows the targeting vector used to replace the 5′ flanking region of the mouse Avpr1a gene with corresponding sequences from the prairie vole. We generated three targeting vectors that were identical except for the microsatellite region they contained, which is indicated by the cross hatched region. Triangles denote loxP sites. (B) shows hybridization of the external Southern probe in correctly targeted recombinants for all three lines. Because the Acc651 site used to screen for recombinant stem cells was located within the floxed region, excision of the NeoR also resulted in the recombinant allele yielding a ∼9.5 kb band when detected with the external probe. (C) shows PCR genotyping. All three lines were backcrossed to a C57Bl/6J background for at least 5 generations prior to neuroanatomical and behavioral experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757045&req=5

pgen-1003729-g002: Targeting vector design.(A) shows the targeting vector used to replace the 5′ flanking region of the mouse Avpr1a gene with corresponding sequences from the prairie vole. We generated three targeting vectors that were identical except for the microsatellite region they contained, which is indicated by the cross hatched region. Triangles denote loxP sites. (B) shows hybridization of the external Southern probe in correctly targeted recombinants for all three lines. Because the Acc651 site used to screen for recombinant stem cells was located within the floxed region, excision of the NeoR also resulted in the recombinant allele yielding a ∼9.5 kb band when detected with the external probe. (C) shows PCR genotyping. All three lines were backcrossed to a C57Bl/6J background for at least 5 generations prior to neuroanatomical and behavioral experiments.
Mentions: The targeting strategy is illustrated in Figure 2. For the meadow line, we screened 288 ES cell clones and identified 1 recombinant. For the prairie short line, 192 clones yielded 2 correct recombinants, and for the prairie long line, 288 clones yielded 2 correct recombinants. This corresponds with an overall recombination efficiency of 0.6% (5 of 768). The floxed PGK-NeoR cassette was successfully removed via breeding to a ubiquitously expressing EIIa-Cre recombinase line as confirmed by PCR and Southern Blot (Figure 2). Because the Acc651 site used to screen for recombinant stem cells was located within the floxed region, excision of the NeoR also resulted in the recombinant allele yielding a ∼9.5 kb band when detected with the external probe. The three resulting recombinant alleles, prairie vole long (pvKI-long), prairie vole short (pvKI-short), or meadow vole (mvKI) were identical in sequence except for the composition of the microsatellite element. All three lines were backcrossed to a C57Bl/6J background for at least 5 generations prior to neuroanatomical and behavioral experiments.

Bottom Line: Previous work has suggested that both the proximal 5' flanking region and a polymorphic microsatellite element within that region of the vole Avpr1a gene are associated with variation in V1a receptor (V1aR) distribution and behavior, but neither has been causally linked.This is the first direct evidence that polymorphic microsatellite elements near behaviorally relevant genes can contribute to diversity in brain gene expression profiles, providing a mechanism for generating behavioral diversity both at the individual and species level.However, our results suggest that many features of species-specific expression patterns are mediated by elements outside of the immediate 5' flanking region of the gene.

View Article: PubMed Central - PubMed

Affiliation: Division of Integrative Neuroscience, Department of Psychiatry, Columbia University, New York, New York, United States of America. zoe.donaldson@gmail.com

ABSTRACT
Certain genes exhibit notable diversity in their expression patterns both within and between species. One such gene is the vasopressin receptor 1a gene (Avpr1a), which exhibits striking differences in neural expression patterns that are responsible for mediating differences in vasopressin-mediated social behaviors. The genomic mechanisms that contribute to these remarkable differences in expression are not well understood. Previous work has suggested that both the proximal 5' flanking region and a polymorphic microsatellite element within that region of the vole Avpr1a gene are associated with variation in V1a receptor (V1aR) distribution and behavior, but neither has been causally linked. Using homologous recombination in mice, we reveal the modest contribution of proximal 5' flanking sequences to species differences in V1aR distribution, and confirm that variation in V1aR distribution impacts stress-coping in the forced swim test. We also demonstrate that the vole Avpr1a microsatellite structure contributes to Avpr1a expression in the amygdala, thalamus, and hippocampus, mirroring a subset of the inter- and intra-species differences observed in central V1aR patterns in voles. This is the first direct evidence that polymorphic microsatellite elements near behaviorally relevant genes can contribute to diversity in brain gene expression profiles, providing a mechanism for generating behavioral diversity both at the individual and species level. However, our results suggest that many features of species-specific expression patterns are mediated by elements outside of the immediate 5' flanking region of the gene.

Show MeSH
Related in: MedlinePlus