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The introduction of supernova system: a vector system for single-cell labeling and gene function analysis
- 1. Division of Neurogenetics (Iwasato lab), National Institute of Genetic, Japan The introduction of Supernova system: a vector system which enables single-cell labeling and labeled cell-specific gene manipulation Upload date: Nov 16th 2016
- 2. We are going to introduce the “Supernova system”, which has been reported in the following two papers: Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo Wenshu Luo*, Hidenobu Mizuno*, Ryohei Iwata, Shingo Nakazawa, Kosuke Yasuda, Shigeyoshi Itohara & Takuji Iwasato** *Co-first authors, ** Correspondence Scientific Reports 6, Article number: 35747 (2016) doi:10.1038/srep35747 NMDAR-Regulated Dynamics of Layer 4 Neuronal Dendrites during Thalamocortical Reorganization in Neonates Hidenobu Mizuno, Wenshu Luo, Etsuko Tarusawa, Yoshikazu M. Saito, Takuya Sato, Yumiko Yoshimura, Shigeyoshi Itohara & Takuji Iwasato** ** Correspondence Neuron, Volume 82, Issue 2, p365–379, 16 April 2014 doi.org/10.1016/j.neuron.2014.02.026
- 3. Background In the mammalian brain, neurons are densely packed and interconnected with each other to form neural circuits that are responsible for higher brain function.
- 4. For understanding the formation and function of the neural circuits, single-cell analysis that dissects connectivity of individual neurons is essential. Dendrite Axon Background In the mammalian brain, neurons are densely packed and interconnected with each other to form neural circuits that are responsible for higher brain function.
- 5. In utero electroporation (IUE), an efficient method for neuronal labeling + - CAG-RFP Embryonic day (E) 13.5 -15.5 E13.5 Deep Layer - +CAG-RFP Labeling cortical neurons L4 L2/3 E14.5 E15.5 Labeling of cortical neurons can be achieved by electroporating a DNA construct into a subpopulation of progenitor cells in the ventricular zone of the embryonic brain. The progenitor cells carrying the DNA undergo neurogenesis, migration, and final differentiation to become mature neurons positioned in distinct cortical layers according to their birth date. Modified from Saito and Nakatsuji, Dev. Biol. (2001) Fukuchi-Shimogori and Grove, Science (2001) Tabata and Nakajima, Neuroscience (2001)
- 6. In utero electroporation (IUE), an efficient method for neuronal labeling Labeling hippocampal neurons E13.5 Deep Layer - +CAG-RFP Labeling cortical neurons E13.5-15.5 Hippocampus -+ CAG-RFP L4 L2/3 E14.5 E15.5 By controlling the direction of electroporation, cells in other brain regions can also be labeled. + - CAG-RFP Embryonic day (E) 13.5 -15.5 Modified from Saito and Nakatsuji, Dev. Biol. (2001) Fukuchi-Shimogori and Grove, Science (2001) Tabata and Nakajima, Neuroscience (2001)
- 7. Electroporation of plasmid pCAG-RFP (CAG-RFP) would label too many cells with high fluorescent intensity. CAG-RFP CAG-GFP (Control) Many Bright
- 8. CAG-Cre CAG-LSL-RFP-WPRE CAG-GFP (Control) CAG-RFP CAG-GFP (Control) Many Bright Sparse Dark For sparse labeling, CAG-Cre in a very low concentration together with pCAG-loxP-STOP- loxP-RFP (CAG-LSL-RFP- WPRE) were often used. In this way, only a small subset of cells is labeled, but mostly remains dark.
- 9. TRE-Cre CAG-LSL-RFP-ires-tTA-WPRE Supernova (Sn) system CAG-GFP (Control) Sparse Bright CAG-Cre CAG-LSL-RFP-WPRE CAG-RFP CAG-GFP (Control) Many Bright Sparse Dark CAG-GFP (Control) Supernova system enables sparse and bright labeling of cortical neurons Mizuno et al., Neuron (2014)
- 10. What is Supernova system and how does it work? Vector 2 CAG Pr XFP pA RT STOP RT ires tTA WPRE Vector 1 SSR pATRE TRE-SSR-WPRE-pA (TRE-SSR) TRE: tetracycline response element; SSR: site-specific recombinase, such as Cre, Flpe and Dre CAG-RT-stop-RT-XFP-ires-tTA-WPRE-pA (CAG-RT-stop-RT-XFP-tTA) RT: recombination target site, such as loxP, FRT, and rox; XFP: fluorescent proteins, such as GFP and RFP; tTA: tetracycline transactivator Vector 2 Vector 1 The elementary composition of IUE-based Supernova includes a set of two vectors:
- 11. pATRE Cre 1 Cre CAG Pr RFP pASTOP loxP ires tTA WPRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE Cre pATRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE No TRE Leakage TRE Leakage loxP Initially, only in a sparse population among many cells that are transfected with both vectors, the leakage of TRE drives above threshold but weak Cre expression. 1 e.g. Cre-based Supernova RFP (Cre-SnRFP) The strategy of Supernova system
- 12. pATRE Cre 1 Cre CAG Pr RFP pAires tTA WPRE tTARFP CAG Pr RFP pA loxP STOP loxP ires tTA WPRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE Cre pATRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE No TRE Leakage TRE Leakage 2 loxP 2 This low level of Cre excises the loxP-stop-loxP cassette in a few copies of CAG-loxP-stop-loxP-RFP-tTA vector, initiating the transcription of RFP and tTA, albeit weakly. The strategy of Supernova system
- 13. tTA CAG Pr RFP pA loxP STOP loxP ires tTA WPRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE loxP pATRE Cre Cre CAG Pr RFP pA loxP STOP loxP ires tTA WPRE Cre pATRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE RFP Negative RFP Positive tTA 3 CAG Pr RFP pAires tTA WPRE tTARFP 3 Through binding with TRE, tTA facilitates further expression of Cre. The strategy of Supernova system
- 14. CAG Pr RFP pA loxP STOP loxP ires tTA WPRE Cre pATRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE CAG Pr RFP pA loxP STOP loxP ires tTA WPRE pATRE Cre Cre CAG Pr RFP pAires tTA WPRE CAG Pr RFP pAires tTA WPRE RFP Negative RFP Positive tTA CAG Pr RFP pAires tTA WPRE tTARFP 4 tTA The strategy of Supernova system Then loxP-stop-loxP cassette is excised from many copies of CAG- loxP-stop-loxP-RFP-tTA vector, and expression of RFP and tTA is increased. This positive loop of tTA/TRE enhancement leads to extremely high levels of expression of both Cre and RFP, only in a small population of transfected cells. 4
- 15. Supernova labeling is sparse and bright enough to visualize the detailed cellular morphologies CAG-GFP Cre-SnRFP Cre-SnRFP P16 50mm Luo et al., Scientific Reports (2016) Cre-SnRFP Dendritic spines Cell body 5mm 50mm Axons 7.5mm Axonal boutons
- 16. Flpe-based and Dre-based Supernova systems also enable bright single-cell labeling Flpe-SnGFP Flpe-SnGFP CAG-LSL-RFP P8 100mm Dre-SnGFP Dre-SnGFP CAG-LSL-RFP P8 100mm CAG Pr GFP pA FRT STOP FRT ires tTA WPRE Flpe/FRT-based Supernova FlpeTRE pAWPRE CAG Pr GFP pA rox STOP rox ires tTA WPRE Dre/rox-based Supernova DreTRE pAWPRE These Supernova vectors can be used along with Cre.
- 17. Supernova is applicable for developmental stages and in adults 2M 1.4% ±0.3% 4M 1.7% ±0.1% P22 1.2% ±0.2% P8 Flpe-SnGFP 1.4% ±0.1% 1.2% ±0.05% 8M 100mm The sparseness and brightness are constant. CAG-RFP Flpe-SnGFP Flpe-SnGFP GFP+ / RFP+ We electroporated Flpe-SnGFP and CAG-RFP into cortex together, then dissected the brains at P8, P22, 2 months (2M), 4M and 8M. Sparseness was evaluated as the ratio of GFP-positive to RFP-positive neurons. As shown above, the ratios and brightness were similar at all ages examined.
- 18. The sparseness of Supernova-labeling is adjustable 0 25 50 75 100 0 1 2 3 45 50 500 (%) No.GFP+/No.RFP+ 50 ng/ml5 ng/ml 500 ng/ml P8 1.4% ±0.1% 48.0% ±5.4% 98.7% ±2.5% 100mm TRE-Flpe CAG-RFP Flpe-SnGFP Flpe-SnGFP GFP+ / RFP+ The sparseness of Supernova labeling is adjustable by simply changing the concentration of TRE-SSR vector concentration in the DNA mixture for IUE. NOTE! Labeling brightness was not altered by changing labeling sparseness.
- 19. Supernova enables co-expression of multiple genes in a single cell CAG Pr GFP pA FRT STOP FRT ires tTA WPRE FlpeTRE pAWPRE CAG Pr RFP pA FRT STOP FRT ires tTA WPRE Flpe-SnRFPFlpe-SnGFP Merge P8 100mm RFP+/GFP+ = 51/53 cells, GFP+/RFP+ = 51/51 cells (n=5 mice) CAG-FSF-GFP-ires-tTA TRE-Flpe CAG-FSF-RFP-ires-tTA Using the Supernova, RFP and GFP were expressed in sparsely labeled neurons with high co-expression efficiency. Flpe-based Supernova vector sets (TRE-Flpe, CAG-FSF-GFP-tTA and CAG-FSF-RFP-tTA) were introduced by IUE.
- 20. Cre-SnnlsRFPCre-SnGFP Merge P4 100mm Cre-SnRFP Cre-SnPSD95-GFP Merge P16 25mm Simultaneous visualization of multiple proteins in a single cell Visualization the cell (GFP) and nucleus (nlsRFP) Visualization the RFP (cell) and PSD95-GFP (individual spines)
- 21. To understand molecular mechanisms operating in individual neurons, a system that enables sparse labeling and labeled cell-specific gene knockout is necessary! Knockout Wild-type Wild-type Wild-type
- 22. Labeled cell-specific gene knockout via Cre-based Supernova CAG Pr RFP pAires tTA WPRE RFP CAG Pr RFP pA loxP STOP loxP ires tTA WPRE Cre pATRE GenomeGenome IUEIUE loxP loxP gene pATRE Cre Cre Flox loxP KO loxP loxP geneFlox Cre RFP Negative RFP Positive Wild-type Knockout (KO)
- 23. Labeled cell-specific gene knockout via Cre-based Supernova CAG Pr RFP pAires tTA WPRE RFP CAG Pr RFP pA loxP STOP loxP ires tTA WPRE Cre pATRE GenomeGenome IUEIUE loxP loxP gene pATRE Cre Cre Flox loxP KO loxP loxP geneFlox Cre RFP Negative RFP Positive Wild-type Knockout (KO) Supernova can achieve sparsely labeled cell-specific gene knockout. • Cre-based Supernova express high level of Cre in Supernova-labeled cells, whereas Cre expression is absent in non-labeled cells. • Thus, a floxed gene in the genome is deleted only in Supernova-labeled cells by Cre expression.
- 24. Labeled cell-specific a2-Chn knockout via Cre-based Supernova 100 mm 500mm CA1 IUE Cre-SnRFP → a2-Chn floxed mouse Merge Cre-SnRFP P14 a2-chimaerin DAPI Cre-SnRFP a2-chimaerin DAPI • For quantitative analysis, we chose α2-chn as a target gene. • We electroporated Cre-SnRFP into the hippocampus of α2- Chn flox/flox mice. The brains were dissected at P14. • As you can see, α2-chimaerin was ubiquitously expressed in CA1, but specifically lacked in SnRFP-labeled neurons.
- 25. Labeled cell-specific a2-Chn knockout via Cre-based Supernova 100 mm 500mm CA1 IUE Cre-SnRFP → a2-Chn floxed mouse No.a2-chimaerin+/No.DAPI+ 0 20 40 60 80 100 1 ** (%) RFP-posi. (Control) (a2-Chn KO) RFP-neg. Merge Cre-SnRFP P14 a2-chimaerin DAPI Cre-SnRFP a2-chimaerin DAPI • We quantified the ratio of α2-chimaerin positive cells in RFP- negative (control) and RFP-positive (SnRFP-labeled) cells. • In CA1 region, 98% of RFP-neg. cells expressed α2-chimaerin, while only 6% of RFP-posi. cells showed α2-chimaerinn signals. • These results demonstrate the high recombination specificity and efficiency of the Supernova-induced gene knockout.
- 26. To achieve single-cell gene manipulation without using floxed mice, we adapted TALEN-based genome editing technology to Supernova. CAG Pr RFP pA FRT STOP FRT ires tTA WPRE Supernova-mediated TALEN FlpeTRE pAWPRE CAG Pr pA FRT STOP FRT TALEN Left CAG-FSF-RFP-ires-tTA TRE-Flpe CAG-FSF-TALEN Left CAG Pr pA FRT STOP FRT TALEN RightCAG-FSF-TALEN Right +
- 27. Labeled cell-specific a2-Chn knockout by Supernova-mediated TALEN Low DAPIFlpe-SnRFP Merge P14a2-chimaerin 20mm 20mm Negative High IUE at E14.5 → wild-type mouse • We electroporated TALEN constructs targeting the endogenous α2-chn together with SnRFP into hippocampus in wild-type mice. • In P14 brain, we divided the cells into three groups depending on intensities of α2-chimaerin signals. There were: a2-chimaerin high, α2- chimaerin negative and α2-chimaerin low cells.
- 28. Labeled cell-specific a2-Chn knockout by Supernova-mediated TALEN a2-Chn-Low a2-Chn-High a2-Chn-Negative Low DAPIFlpe-SnRFP Merge P14a2-chimaerin 20mm 20mm Negative High 0 20 40 60 80 100 1 2 RFP+RFP- Cellpercentage (%) IUE at E14.5 → wild-type mouse • We found that 95% of RFP-negative cells were α2-chimaerinhigh cells. • In contrast, 76% of RFP-positive cells were α2-chimaerinnegative cells. The rest were α2-chimaerinlow cells, whereas there were no α2- chimaerinhigh cells.
- 29. Labeled cell-specific a2-Chn knockout by Supernova-mediated TALEN Low DAPIFlpe-SnRFP Merge P14a2-chimaerin 20mm 20mm Negative High 0 20 40 60 80 100 1 2 RFP+RFP- Cellpercentage (%) IUE at E14.5 → wild-type mouse These results suggest that Supernova- mediated TALEN successfully inhibited α2-chimaerin expression in the hippocampal neurons of wild-type mice. a2-Chn-Low a2-Chn-High a2-Chn-Negative
- 30. Labeled cell-specific a2-Chn editing by Supernova-mediated TALEN Sampling at P1 Cloning Sequencing PCR (13 clones) 1 : No-mutation IUE at E14.5 Collecting labeled cells by FACS Wild type Mutations 5’- 3’- -3’ -5’ Left TALEN Right TALEN 17bp a2-Chn genome 12: Edited • We amplified the target genome locus from a pool of 200 FACS-sorted cells using PCR and cloned into the plasmids. • In 13 clones sequenced, 12 had mutations (three patterns) and one had wild- type sequence. 9 mutation patterns in total were identified.
- 31. Supernova-mediated CRISPR CAG-LSL-GFP-ires-tTA TRE-Cre CAG Pr GFP pA loxP STOP loxP ires tTA WPRE CreTRE pAWPRE U6-gRNA-CAG-LSL-Cas9 U6 Pr sgRNA CAG Pr loxP STOP loxP hSpCas9 pA + We also combined another genome editing technology CRISPR/Cas9 with Supernova.
- 32. Labeled cell-specific Creb knockout by Supernova-mediated CRISPR/Cas9Control P8 SnCRISPR-Creb 50mm Cre-SnGFP Cre-SnGFP/CREB/DAPICREB P8 50mm Control vector: without targeting sequence in sgRNA IUE at E14.5 → wild-type mouse • To evaluate the effectiveness, We chose Creb1 as the target gene because of its strong and ubiquitous expression in the hippocampus. • We electroporated CRISPR/Cas9 constructs together with SnGFP into the hippocampus in wild-type mice and examined CREB protein expression in the P8 brain.
- 33. Control P8 SnCRISPR-Creb 50mm Cre-SnGFP Cre-SnGFP/CREB/DAPICREB P8 50mm IUE at E14.5 → wild-type mouse • We observed that, when the control vector (without targeting sequence in sgRNA) was used, all GFP-positive neurons expressed CREB. • In contrast, when the vectors carrying sgRNA-targeting Creb1 were transfected, CREB expression was undetectable in almost all GFP-positive neurons. Labeled cell-specific Creb knockout by Supernova-mediated CRISPR/Cas9 Control vector: without targeting sequence in sgRNA
- 34. Control P8 SnCRISPR-Creb 50mm Cre-SnGFP Cre-SnGFP/CREB/DAPICREB P8 50mm 0 20 40 60 80 100 1 2 No.CREB+/No.GFP+ (%) SnCRISPR-Creb (n=85 cells) Control (n=111 cells) IUE at E14.5 → wild-type mouse Labeled cell-specific Creb knockout by Supernova-mediated CRISPR/Cas9 Control vector: without targeting sequence in sgRNA
- 35. (12 clones) 12: Edited 0: wild-type Mutations Wild type PAM Deletion: 5 patterns sgRNA-Creb1 Mutations Wild type PAM sgRNA-Creb1Insertion: 2 patterns Sampling at P1 Cloning Sequencing PCRIUE at E14.5 Collecting labeled cells by FACS • We also confirmed the Creb1 editing in GFP-positive cells through FACS followed by sequencing analysis on a pool of 300 cells. • We identified seven patterns of mutations in all 12 clones obtained. • No wild-type clone was identified. Labeled cell-specific Creb knockout by Supernova-mediated CRISPR/Cas9
- 36. Supernova-mediated RNAi CAG-LSL-XFP-ires-tTA TRE-Cre CAG Pr XFP pA loxP STOP loxP ires tTA WPRE CreTRE pAWPRE CAG-LSL-mir30 (RNAi) CAG Pr pA loxP STOP loxP mir30 (RNAi) + We also combined the Supernova with RNA interference (RNAi).
- 37. GFP Control GFP_RNAi LacZ LacZ_RNAiControl 50mm 50mm CAG-CAT-GFP reporter mouse Rosa-LSL-LacZ reporter mouse GFPintensity Control RNAi *** LacZintensity *** Control RNAi By IUE of Supernova-mediated expression vectors carrying shRNA against target genes, we efficiently reduced their expression level in sparsely labeled cortical neurons.
- 38. Supernova-mediated single-cell labeling in vivo: Summary • Single cell labeling shows high fluorescent intensity with essentially no background • Labeling sparseness and brightness are constant from early postnatal stages to adulthood • Labeling sparseness is adjustable • Simultaneous expression of multiple genes in a single-cell is possible Labeled cell-specific gene manipulation in vivo by Supernova: Genome editing by Sn-TALEN Genome editing by Sn-CRISPR/Cas9 Gene knockdown by Sn-RNAi in floxed mice in wild-type mice• Single cell- • Single cell- Gene knockout by Cre-Supernova
- 39. AAV-based Supernova system AAV-EF1a-DIO-ｔTA-RFP AAV-TRE-Cre CreTRE pAWPRE R-ITRL-ITR EF1a Pr RFP pA loxP P2A loxP tTA WPRE R-ITRL-ITR lox2722 lox2722 We also developed the AAV-based Supernova system! Besides IUE, Virus vector-mediated gene delivery is another powerful approach for gene expression in vivo. Therefore, we also developed Adeno-associated virus (AAV) –based Supernova system. However, due to the limitation of insert size of the AAV vector (<5kb), the loxP-STOP-loxP cassette used in IUE-based Supernova vectors could not be used. Instead, we used the strategy of double-floxed inverted open reading frame (DIO).
- 40. AAV-EF1a-GFP AAV-SnRFP AAV-SnRFP AAV-based Supernova labeling is sparse and bright enough to visualize the detailed cellular morphologies 500mm 100mm 10mm
- 41. AAV-SnRFP LacZ Merge CA1 Cortex • To examine the efficiency and specificity of Cre-mediated genomic DNA recombination, we injected AAV-SnRFP into hippocampal CA1 regions of Rosa26-loxP-stop-loxP-nlsLacZ (RNZ) reporter mice at P10, and brains were sampled at 40DPI. • We observed that almost all RFP-labeled neurons expressed LacZ (76/80cells, n = 3 mice). Moreover, all LacZ-positive neurons were labeled by AAV-SnRFP (76/76 cells, n = 3 mice). Similar results were obtained in the cortex (bottom panel). In AAV-based Supernova system, a high level of Cre is expected to be expressed only in sparsely labeled neurons. 50mm 50mm
- 42. AAV-SnRFP was injected into the hippocampus of α2-Chn flox/flox mice at P2, and brains were dissected at P18 (16DPI). In the hippocampal CA1, 97% of RFP-negative cells expressed α2- chimaerin (204/210 cells, n = 3 mice), while none of RFP-positive neurons showed α2-chimaerin signals (0/20 cells, n = 3 mice). DAPI a2-chimaerin AAV-SnRFP Merge CA1 High efficiency of AAV-Supernova system in labeled cell-specific gene knockout in floxed mice.
- 43. AAV-based Supernova system AAV-EF1a-DIO-ｔTA-RFP AAV-TRE-Cre CreTRE pAWPRE R-ITRL-ITR EF1a Pr RFP pA loxP P2A loxP tTA WPRE R-ITRL-ITR lox2722 lox2722 Supernova series of vector systems (both IUE- and AAV-based systems) are useful systems that enable both sparse cell-labeling with high fluorescence intensity and labeled cell-specific gene manipulation. CAG-RT-stop-RT-XFP-tTA CAG Pr XFP pA RT STOP RT ires tTA WPRE TRE-SSR SSR pATRE IUE-based Supernova system Supernova based on two gene delivery systems
- 44. Supernova for your research • Iwasato laboratory web site https://www.nig.ac.jp/labs/NeurGen/ • Supernova support site http://snsupport.webcrow.jp/ Plasmids will be available from Addgene. Check the support site!
- 45. Acknowledgements We thank all the co-authors of H. Mizuno, et al., Neuron 2014 and W. Luo, et al., Sci. Rep. 2016 for their contributions on developing Supernova systems. We also appreciate Yuka Ryomoto for valuable advices on information disclosure. Contributions to preparing the SlideShare presentation Wenshu Luo, Shingo Nakazawa Ramasamy Kandasamy, Hidenobu Mizuno, Takuji Iwasato Slides production Offering comments and suggestions Contact: Takuji Iwasato <tiwasato nig.ac.jp> Division of Neurogenetics (Iwasato lab), National Institute of Genetics; Department of Genetics, SOKENDAI (The Graduate University for Advanced Studies) @