Characterizations of Hirudo medicinalis DNA promoters for targeted gene expression

Baker, Maureen; Macagno, Eduardo R.

doi:10.1016/j.jneumeth.2006.02.026

Cited by 16 publications

(11 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Small molecule indicators have a distinct advantage over protein-based indicators because of the difficulty of transfecting adult leech neurons. 34 We prepared desheathed mid-body ganglia from H. medicinalis stained with VF2.1(OMe).H (200 nM, 20 min.). Following treatment with VF2.1(OMe).H, cellular membranes in the ganglia showed strong fluorescence characteristic of VF dyes (Fig 5a).…”

Section: Resultsmentioning

confidence: 99%

Improved PeT Molecules for Optically Sensing Voltage in Neurons

Woodford¹,

Frady²,

et al. 2015

View full text Add to dashboard Cite

VoltageFluor (VF) dyes have the potential to optically measure voltage in excitable membranes with the combination of high spatial and temporal resolution essential to better characterize the voltage dynamics of large groups of excitable cells. VF dyes sense voltage with high speed and sensitivity using photoinduced electron transfer (PeT) through a conjugated molecular wire. We show that tuning the driving force for PeT (ΔGPeT + w) through systematic chemical substitution modulates voltage sensitivity, estimate (ΔGPeT + w) values from experimentally measured redox potentials, and validate the voltage sensitivities in patch-clamped HEK cells for 10 new VF dyes. VF2.1(OMe).H, with a 48% ΔF/F per 100 mV, shows approximately 2-fold improvement over previous dyes in HEK cells, dissociated rat cortical neurons, and medicinal leech ganglia. Additionally, VF2.1(OMe).H faithfully reports pharmacological effects and circuit activity in mouse olfactory bulb slices, thus opening a wide range of previously inaccessible applications for voltage sensitive dyes.

show abstract

Section: Resultsmentioning

confidence: 99%

Improved PeT Molecules for Optically Sensing Voltage in Neurons

Woodford¹,

Frady²,

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Full-length clones of Hve-inx1, Hve-inx2, Hve-inx6, and Hve-inx14 (GenBank accession numbers AJ512833, AJ512834, DQ228703, and JQ231020.1, respectively) were generated by PCR and subcloned into a modified EGFP-N1 (Clontech) expression vector in which the CMV promoter was excised and the promoter of a leech cytoplasmic actin gene, HmAct1 (GenBank accession number DQ333328) inserted in its place (Baker and Macagno, 2006). Cellular transformation was performed using a gene gun to deliver plasmid-covered gold particles (Shefi et al, 2006), or by direct intranuclear injection in the intact developing embryo (Baker and Macagno, 2006).…”

Section: Methodsmentioning

confidence: 99%

Ectopic Expression of Select Innexins in Individual Central Neurons Couples Them to Pre-Existing Neuronal or Glial Networks That Express the Same Innexin

et al. 2012

View full text Add to dashboard Cite

Fifteen of the 21 innexin (Inx) genes (Hve-inx) found in the genome of the medicinal leech, Hirudo verbana, are expressed in the CNS (Kandarian et al., 2012). Two are expressed pan-neuronally, while the others are restricted in their expression to small numbers of cells, in some cases reflecting the membership of known networks of electrically coupled and dye-coupled neurons or glial cells. We report here that when Hve-inx genes characteristic of discrete coupled networks were expressed ectopically in neurons known not to express them, the experimental cells were found to become dye coupled with the other cells in that network. Hve-inx6 is normally expressed by only three neurons in each ganglion, which form strongly dye-coupled electrical connections with each other [Shortening-Coupling interneuron (S-CI) network] (Muller and Scott, 1981; Dykes and Macagno, 2006). But when Hve-inx6 was ectopically expressed in a variety of central embryonic neurons, those cells became dye coupled with the S-CI network. Similarly, Hve-inx2 is normally uniquely expressed by the ganglion’s large glial cells, but when it was ectopically expressed in different central neurons, they became dye coupled to the glial cells. In contrast, overexpression of the pan-neuronal Inx genes Hve-inx1 and Hve-inx14 did not yield any novel instances of dye coupling to pre-existent neuronal networks. These results reveal that expression of certain innexins is sufficient to couple individual neurons to pre-existing networks in the CNS. We propose that a primary determinant of selective neuronal connectivity and circuit formation in the leech is the surface expression of unique subsets of gap junctional proteins.

show abstract

“…Plasmid injections into nuclei of post-mitotic cells have been used in late stage embryos of the medicinal leech Hirudo to express reporter constructs as well as for functional studies, (Baker and Macagno, 2006; Shefi et al, 2006; Baker and Macagno, 2007; Baker et al, 2008). Previous attempts to drive ectopic gene expression in the early Helobdella embryo using plasmid injections were problematic, however; expression was highly mosaic (i.e., was seen in only a few of the progeny of the injected cell) and the injected lineage developed abnormally independent of the expression of the transgene…”

Section: Introductionmentioning

confidence: 99%

High resolution cell lineage tracing reveals developmental variability in leech

Gline¹,

Kuo²,

Stolfi³

et al. 2009

Developmental Dynamics

View full text Add to dashboard Cite

Knowing the normal patterns of embryonic cell proliferation, migration and differentiation is a cornerstone for understanding development. Yet for most species, the precision with which embryonic cell lineages can be determined is limited by technical considerations (the large numbers of cells, extended developmental times, opacity of the embryos), and these are exacerbated by the inherent variability of the lineages themselves. Here we present an improved method of cell lineage tracing in the leech Helobdella, driving the expression of a nuclearly localized histone H2B:GFP fusion protein in selected lineages by microinjection of a plasmid vector. This construct generates a long lasting and minimally mosaic signal with single cell resolution, and does not disrupt the development of most lineages tested. We have validated this technique by elucidating details of cell lineages contributing to segmental and prostomial tissues that could not be observed with standard dextran lineage tracers.

show abstract

Characterizations of Hirudo medicinalis DNA promoters for targeted gene expression

Cited by 16 publications

References 38 publications

Improved PeT Molecules for Optically Sensing Voltage in Neurons

Improved PeT Molecules for Optically Sensing Voltage in Neurons

Ectopic Expression of Select Innexins in Individual Central Neurons Couples Them to Pre-Existing Neuronal or Glial Networks That Express the Same Innexin

High resolution cell lineage tracing reveals developmental variability in leech

Contact Info

Product

Resources

About