Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun

Wellmann, Henning; Kaltschmidt, Barbara; Kaltschmidt, Christian

doi:10.1016/s0165-0270(99)00094-1

Cited by 68 publications

(50 citation statements)

References 23 publications

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“…Biolistic siRNA delivery was performed in 14 DIV slice cultures with either a nontargeting siRNA (siRNA-CTL), or siRNA directed against Stau1 (siRNA-STAU1) or Stau2 (siRNA-STAU2-1). While biolistic DNA plasmid transfection in organotypic slice cultures leads to only a small percentage of transfected neurons (1%-2%) (Wellmann et al 1999;Boda et al 2004;Govek et al 2004), we have found that delivery of siRNAs is much more efficient (Lebeau et al 2008). Indeed, using a fluorescently labeled siRNA, one can detect high levels of siRNA in most of the superficial principal neurons in slices where the electrophysiological recordings are performed (Lebeau et al 2008).…”

Section: Down-regulation Of Stau2 By Sirna Transfectionmentioning

confidence: 84%

Staufen 2 regulates mGluR long-term depression and Map1b mRNA distribution in hippocampal neurons

Lebeau¹,

Miller²,

Tartas³

et al. 2011

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The two members of the Staufen family of RNA-binding proteins, Stau1 and Stau2, are present in distinct ribonucleoprotein complexes and associate with different mRNAs. Stau1 is required for protein synthesis -dependent long-term potentiation (L-LTP) in hippocampal pyramidal cells. However, the role of Stau2 in synaptic plasticity remains unexplored. We found that unlike Stau1, Stau2 is not required for L-LTP. In contrast, Stau2, but not Stau1, is necessary for DHPG-induced protein synthesis-dependent long-term depression (mGluR-LTD). While Stau2 is involved in early development of spines, its down-regulation does not alter spine morphology or spontaneous miniature synaptic activity in older cultures where LTD occurs. In addition, Stau2, but not Stau1, knockdown reduces the dendritic localization of Map1b mRNA, a specific transcript involved in mGluR-LTD. Moreover, mGluR stimulation with DHPG induces Map1b, but not Map2, mRNA dissociation from mRNA granules containing Stau2 and the ribosomal protein P0. This dissociation was not observed in cells in which Stau2 was depleted. Finally, Stau2 knockdown reduces basal Map1b protein expression in dendrites and prevents DHPG-induced increases in dendritic Map1b protein level. We suggest a role for Stau2 in the generation and regulation of Map1b mRNA containing granules that are required for mGluR-LTD.

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Section: Down-regulation Of Stau2 By Sirna Transfectionmentioning

confidence: 84%

Staufen 2 regulates mGluR long-term depression and Map1b mRNA distribution in hippocampal neurons

Lebeau¹,

Miller²,

Tartas³

et al. 2011

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“…PCs in organotypic cultures are well known to be resistant to classical transfection methods, and nonviral vectors such as the gene gun are not efficient enough to allow the transfection of a high proportion of PCs in slices (Lo et al, 1994;Wellmann et al, 1999;Murphy and Messer, 2001;Ghoumari et al, 2002). In contrast, lentiviral vectors are capable of transducing cells that are dividing, growth arrested, or postmitotic (Poeschla et al, 1998) and have been shown previously to efficiently transduce Purkinje Transduced PCs were identified by GFP expression for Lenti-GFP-infected slices (A) and by ROR␣ nuclear immunolabeling for Lenti-hROR␣1-infected slices (B): these PCs display a similar identified dendritic tree.…”

Section: Discussionmentioning

confidence: 99%

Retinoid-Related Orphan Receptor α Controls the Early Steps of Purkinje Cell Dendritic Differentiation

et al. 2006

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Dendritic differentiation involves both regressive and growth events. The mechanisms controlling the regressive events are poorly understood. This study is aimed at determining the role of the nuclear receptor retinoid-related orphan receptor ␣ (ROR␣) in Purkinje cell (PC) dendritic differentiation in organotypic cultures. As observed in vivo, in these cultures, fusiform PCs with embryonic bipolar shape undergo regression before the outgrowth of the ultimate dendritic tree. We show that lentiviral-mediated hROR␣1 overexpression in fusiform PCs leads to a cell-autonomous accelerated progression of dendritic differentiation. In addition, ROR␣ is necessary for the PC regressive events: whereas staggerer ROR␣-deficient PCs remain in the embryonic fusiform stage, replacement of hROR␣1 restores normal dendritogenesis. These results demonstrate that ROR␣ expression in fusiform PCs is crucial for the dendritic regression and progression of the following step of extension of dendritic processes. However, it does not seem to participate to the last stage of dendritic growth. This study identifies ROR␣ as a nuclear receptor crucial for the control of dendritic remodeling during development.

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“…Rac40 was made with PCR sitedirected mutagenesis. The standard calcium phosphate precipitation method was used to transfect neurons at 5-7 DIV (Liao et al, 2005), whereas the gene gun technique was used to transfect neurons at 21 DIV (Wellmann et al, 1999). The culture dishes fit tightly in a homemade holding chamber on a fixed platform above an inverted microscope sitting on an X-Y translation stage (Burleigh Instruments, Fishers, NY).…”

Section: Methodsmentioning

confidence: 99%

Rac1 Induces the Clustering of AMPA Receptors during Spinogenesis

Wiens¹,

Lin²,

Liao³

2005

J. Neurosci.

114

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Glutamatergic synapses switch from nonspiny synapses to become dendritic spines during early neuronal development. Here, we report that the lack of sufficient Rac1, a small RhoGTPase, contributes to the absence of spinogenesis in immature neurons. The overexpression of green fluorescence protein-tagged wild-type Rac1 initiated the formation of dendritic spines in cultured dissociated hippocampal neurons younger than 11 d in vitro, indicating that Rac1 is likely one of the missing pieces responsible for the lack of spines in immature neurons. The overexpression of wild-type Rac1 also induced the clustering of AMPA receptors (AMPARs) and increased the amplitude of miniature EPSCs (mEPSCs). The expression of constitutively active Rac1 induced the formation of unusually large synapses with large amounts of AMPAR clusters. Also, our live imaging experiments revealed that the contact of an axon induced the clustering of Rac1, and subsequent morphological changes led to spinogenesis. Additionally, the overexpression of wild-type Rac1 and constitutively active Rac1 increased the size of preexisting spines and the amplitude of mEPSCs in mature neurons (Ͼ21 d in vitro) within 24 h after transfection. Together, these results indicate that activation of Rac1 enhances excitatory synaptic transmission by recruiting AMPARs to synapses during spinogenesis, thus providing a mechanistic link between presynaptic and postsynaptic developmental changes. Furthermore, we show that Rac1 has two distinct roles at different stages of neuronal development. The activation of Rac1 initiates spinogenesis at an early stage and regulates the function and morphology of preexisting spines at a later stage.

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Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun

Cited by 68 publications

References 23 publications

Staufen 2 regulates mGluR long-term depression and Map1b mRNA distribution in hippocampal neurons

Staufen 2 regulates mGluR long-term depression and Map1b mRNA distribution in hippocampal neurons

Retinoid-Related Orphan Receptor α Controls the Early Steps of Purkinje Cell Dendritic Differentiation

Rac1 Induces the Clustering of AMPA Receptors during Spinogenesis

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