29 research outputs found
Specificity of EGFP expression from the p563 AAV construct.
<p>Immunohistochemistry was performed two weeks after injection of the AAVs into the rat SON. Brain sections in A and D show EGFP fluorescence, in B, PS38 antibody staining, in C, merged EGFP fluorescence and PS38 staining, in E, PS41 antibody staining, and in F, merged EGFP fluorescence and PS41 staining. Note that while the EGFP is abundantly expressed in the OXT MCNs (panel C), there is virtually no expression of the EGFP in the AVP MCNs (panel F). Abbreviations: OC, optic chiasm. Scale line in F is the same for all panels.</p
Description of AAVs containing OXT promoter deletion constructs used in this study.
<p>A. Shows plasmid pFBGR, which was used for making all constructs, this plasmid contains the AAV left and right inverted terminal repeats (ITR). B. shows diagrams of constructs made containing sequences 568, 440, 325, 216, 100, and 50 bp upstream of the transcription start site of the OXT gene. These constructs contained all the three exons and two introns in the OXT gene and with the EGFP reporter inserted at the end of the coding region of exon III, followed by 768 bp downstream of OXT exon III. All constructs were placed into pFBGR between the XbaI sites after removal of the 2291 bp XbaI band (see panel A).</p
Diagram summarizing the cis-regulatory domains in the oxytocin gene promoter as suggested by the data in this study.
<p>The β50 bp and β100 bp OXT promoter domains are sufficient to produce expression of an EGFP reporter in both OXT and AVP MCNs in the SON, and in a population of smaller neurons just dorsal to the SON. The β100 to β216 bp domain appears to contain a repressor element that inhibits expression specifically in AVP MCNs. The β216 to β325 domain appears to repress expression in an unidentified dorsal neuron population above the SON, and the β216 to β440 bp domain may have enhancer elements which are operative in the OXT MCNs.</p
Analysis of the specificity of expression of EGFP from AAVs containing various promoter deletion constructs (shown in <b>Fig. 1B</b>) two weeks after their injection into rat SONs.
<p>The specific promoter lengths that were injected into the SONs in each of the experiments is shown in the lower left of the panels. Immunohistochemical analyses show that each of the oxytocin promoter AAV constructs was selectively expressed in oxytocin cells. Each panel illustrates merged images of AVP-neurophysin (PS 41) immunoreactivity shown as red, and the EGFF expression shown as green. Note that while the EGFP is clearly not expressed in the AVP MCNs in panels AβD where the promoter constructs contain 216 bp or more of the upstream region, the p100 construct (panel E) and p50 construct (Panel F) shows EGFP expression in both OXT. Hence the key elements regulating of the selectivity of OXT gene expression must reside between in the β216 to β100 bp domain in the promoter. Abbreviations: OC, optic chiasm. The 100 Β΅m scale line in lower right is the same for all panels.</p
Efficacy of targeting both OXT and AVP magnocellular neurons (MCNs) in rat supraoptic nucleus (SON) by stereotaxic injections of AAVs.
<p>The AAVs used were chimeric AAV6 serotypes with an AAV2 ITR and an AAV6 capsid (see Methods). A. Illustrates a rat brain coronal section showing the placement of 30 gauge needles used for bilateral injections of AAVs to target the SON. Stereotaxic coordinates are presented in Methods. Panels BβD show the results of injecting an AAV containing the CMV promoter fused to an EGFP reporter into the rat SON. B. Illustrates EGFP fluorescence in the MCNs of the SON found two weeks after injection. C. shows OXT MCNs in the SON after staining with PS 38 antibody (an OXT MCN marker). D. Shows merged EGFP fluorescence and PS 38 staining. White Arrows in panels BβD show MCNs in the SON that express EGFP co-localized with PS 38-immunoreactivity. Yellow arrow in panels BβD show MCNs that express EGFP which is not co-localized with PS 38-immunoreactivity, and hence are presumptive AVP MCNs. Abbreviations in A: cc, corpus callosum; LV, lateral ventricle; f, fornix; 3 V, third ventricle; ic, internal capsule; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; OC, optic chiasm. Scale line in D is the same for B and C.</p
Mouse vasopressin deletion constructs and plasmid that were used to make the recombinant AAV (rAAV) vectors.
<p>A. All vasopressin constructs contained an EGFP reporter fused to exon I, as well as the endogenous poly-A signal found at the untranslated region of exon III, and an additional 173 bases that follow exon III. The vasopressin promoter region was systematically reduced such that promoter lengths tested were: 2.0 kbp, 1.5 kbp, 950 bp, 543 bp, 421 bp, 288 bp, 116 bp and 50 bp upstream of the transcription start site. B. Illustration of the AAV plasmid into which the assorted sequences described in A were inserted is shown. Abbreviations: ITR, inverted terminal repeat; 3β² UTR, 173 untranslated bases that follow exon III.</p
Diagram illustrating the cis-regulatory domains in the vasopressin gene promoter as suggested by the data in this study.
<p>Since the 288 VPI.EGFP construct still maintains the vasopressin MCN specific expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone-0048860-g006" target="_blank">Fig. 6F</a>) we hypothesize that there is an activator of expression in the Avp MCNs and a possible repressor element with an upper boundary of β288 kbp that inhibits expression selectively in the Oxt MCNs. We further propose that there are at least three enhancer domains upstream of the transcription start site of the Avp gene that are involved in expression in the Avp MCNs, which we designate enhancer 3 located between β2 kbp to β1.5 kbp, enhancer 2 located between β1.5 kbp to β950 bp, and enhancer 1 located between β543 bp to β288 bp (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone-0048860-g004" target="_blank">Fig. 4</a>). The β288 to 116 bp region appears to contain the cell-type specific element(s) (see text), and an osmotically-responsive element also appears to be present within the 288 bp construct (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone.0048860.s003" target="_blank">Fig. S3</a>).</p
Cell-Type Specific Expression of the Vasopressin Gene Analyzed by AAV Mediated Gene Delivery of Promoter Deletion Constructs into the Rat SON In Vivo
<div><p>The magnocellular neurons (MCNs) in the supraoptic nucleus (SON) of the hypothalamus selectively express either oxytocin (Oxt) or vasopressin (Avp) neuropeptide genes. In this paper we examine the cis-regulatory domains in the Avp gene promoter that are responsible for its cell-type specific expression. AAV vectors that contain various Avp gene promoter deletion constructs using EGFP as the reporter were stereotaxically injected into the rat SON. Two weeks following the injection immunohistochemical assays of EGFP expression from these constructs were done to determine whether the expressed EGFP reporter co-localizes with either the Oxt- or Avp-immunoreactivity in the MCNs. The results identify three major enhancer domains located at β2.0 to β1.5 kbp, β1.5 to β950 bp, and β950 to β543 bp in the Avp gene promoter that regulate the expression in Avp MCNs. The results also show that cellβtype specific expression in Avp MCNs is maintained in constructs containing at least 288 bp of the promoter region upstream of the transcription start site, but this specificity is lost at 116 bp and below. Based on these data, we hypothesize that the β288 bp to β116 bp domain contains an Avp MCN specific activator and a possible repressor that inhibits expression in Oxt-MCNs, thereby leading to the cell-type specific expression of the Avp gene only in the Avp-MCNs.</p> </div
Analysis of the selectivity of expression of EGFP in MCNs in the SON after injections of rAAVs containing the 50 bp promoter deletion construct.
<p>Panels A and D illustrate the EGFP immunoreactivity (EGFP-ir) only, B shows the Avp marker (PS41-ir) only, E shows the Oxt marker (PS38) only. The merged views of the EGFP-ir with either the Avp marker (PS41-ir in C) or the Oxt marker (PS38-ir in F) are shown. Examples of MCNs with colocalized EGFP-ir and PS38-ir or PS41-ir are depicted by yellow arrows and the white arrows show cells containing EGFP-ir only. The EGFP-ir was detected using 33-second photographic exposures as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone-0048860-g005" target="_blank">Fig. 5</a>. Scale bar in panel D is 100 Β΅m, and is the same for all the panels. Abbreviations: OC, optic chiasm.</p
Stereotaxic microinjection of AAV vectors into the supraoptic nuclei (SONs) of adult male rats.
<p>A. Illustrates the coordinates of the injections relative to bregma: β1.3 mm caudal, 1.8 mm lateral (left and right), and β8.8 mm ventral. A total volume of 3 Β΅l of vector was injected at a convection-enhanced delivery rate of 0.3 Β΅l/minute, which resulted in vector completely filling the SON (see inset in A). B. Shows a view of a unilateral SON in a coronal section of the rat hypothalamus after injection into the SON of a rAAV containing the pan-specific CMV promoter fused to an EGFP reporter. Note that the MCNs in the SON show intense EGFP fluorescence and that areas dorsal to the SON also show significant although much less dense cellular fluorescence. This illustrates the total area of transduction deriving from this AAV injection. Asterisk shows the estimated position of the tip of the injection needle. The white dotted line borders the dorsal side of the SON. Scale line represents 100 Β΅m. Abbreviations: OC, optic chiasm; SON, supraoptic nucleus.</p