Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo
Open accessAbstract
It is becoming increasingly clear that single cortical neurons encode complex and behaviorally relevant signals, but efficient means to study gene functions in small networks and single neurons in vivo are still lacking. Here, we establish a method for genetic manipulation and subsequent phenotypic analysis of individual cortical neurons in vivo . First, lentiviral vectors are used for neuron-specific gene delivery from α-calcium/calmodulin-dependent protein kinase II or Synapsin I promoters, optionally in combination with gene knockdown by means of U6 promoter-driven expression of short-interfering RNAs. Second, the phenotypic analysis at the level of single cortical cells is carried out by using two-photon microscopy-based techniques: high-resolution two-photon time-lapse imaging is used to monitor structural dynamics of dendritic spines and axonal projections, whereas cellular response properties are analyzed electrophysiologically by two-photon microscopydirected whole-cell recordings. This approach is ideally suited for analysis of gene functions in individual neurons in the intact brain.
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Conventionally used AAV backbones with a 1.3 kb CaMKIIα promoter, WPRE, and human growth hormone polyadenylation signal have a capacity of 2.4 kb.
“Conventionally used AAV backbones which include a commonly used neuronal expression cassette composed of 1.3 kb CaMKIIα promoter (which shows comparable expression efficiency with 0.4 kb CaMKIIα promoter [10] used in CWB), WPRE, and human growth hormone polyadenylation signal [23-25] has a capacity of 2.4 kb.”
From Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons (2014) · cites this paper as [10]
Table 1 (Methods, p. 2) identifies a 1.3-kb α-CaMKII promoter used in this paper’s lentiviral vectors. The Discussion (p. 6) gives an approximately 10-kb limit for a lentiviral promoter-and-gene cassette, not a 2.4-kb capacity for an AAV backbone. The cited passages do not establish the claimed AAV cassette with WPRE and a human growth hormone polyadenylation signal. (LLM, conf=0.89)
Experimental — accuracy not yet measuredThe 1.3 kb CaMKIIα promoter shows comparable expression efficiency with the 0.4 kb CaMKIIα promoter.
“which shows comparable expression efficiency with 0.4 kb CaMKIIα promoter [10] used in CWB”
From Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons (2014) · cites this paper as [10]
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- Authors
- Dittgen, T., Nimmerjahn, A., Komai, S., Licznerski, P., Waters, J., Margrie, T. W., Helmchen, F., Denk, W., Brecht, M., & Osten, P.
- Affiliations
- Max Planck Institute for Medical Research (DE)
- ORCID
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2004-12-17
- Volume
- 101
- Issue
- 52
- Pages
- 18206-18211
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC539748
- OpenAlex
- W2064886074
- Cited by
- 485
- References
- 6
- Citations by year
- 2026: 19 · 2025: 11 · 2024: 12 · 2023: 13 · 2022: 20 · 2021: 20 · 2020: 21 · 2019: 24 · 2018: 22 · 2017: 25 · 2016: 24 · 2015: 24 · 2014: 26 · 2013: 29 · 2012: 30
- FWCI
- 6.46
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Cold Spring Harbor Laboratory Institutional Repository (Cold Spring Harbor Laboratory) (acceptedVersion)
- Max Planck Institute for Plasma Physics (submittedVersion)
- UCL Discovery (University College London) (submittedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Topics
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- lentiviral vectors, short interfering RNA knockdown, dendritic spine dynamics, axonal projections, in vivo electrophysiology
- MeSH terms
- Animals, Electrophysiology, Genetic Vectors, Hippocampus, Image Processing, Computer-Assisted, Mice, Inbred C57BL, Microscopy, Neurons, Phenotype, Promoter Regions, Genetic, Recombinant Proteins, RNA, Small Nuclear, Lentivirus, Synapsins, Photons, Calcium-Calmodulin-Dependent Protein Kinases, RNA, Small Interfering, Green Fluorescent Proteins, Mice, Calcium-Calmodulin-Dependent Protein Kinase Type 2
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