The introduction of supernova system: a vector system for single-cell labeling and gene function analysis

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

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

http://www.cell.com/neuron/abstract/S0896-6273(14)00157-3

http://www.nature.com/articles/srep35747



BackgroundIn the mammalian brain,

neurons are densely packed and interconnected with each other

to form neural circuits that are responsible for higher brain function.

For understanding the formation and function of the neural circuits,

single-cell analysis that dissects connectivity of individual neurons

is essential.

Dendrite

Axon

BackgroundIn the mammalian brain,

neurons are densely packed and interconnected with each other

to form neural circuits that are responsible for higher brain function.

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)

In utero electroporation (IUE), an efficient method for neuronal labeling

Labeling hippocampal neurons

E13.5

Deep Layer

-

+CAG-RFP

Labeling cortical neurons

E13.5-15.5Hippocampus

-+

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 -15.5Modified from

Saito and Nakatsuji, Dev. Biol. (2001)

Fukuchi-Shimogori and Grove, Science (2001)

Tabata and Nakajima, Neuroscience (2001)

Electroporation of plasmidpCAG-RFP (CAG-RFP) wouldlabel too many cells withhigh fluorescent intensity.

CAG-RFP

CAG-GFP (Control)

Many

Bright

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.

TRE-Cre

CAG-LSL-RFP-ires-tTA-WPRE

Supernova (Sn) system

CAG-GFP (Control)

Sparse

Bright

CAG-Cre

CAG-LSL-RFP-WPRECAG-RFP

CAG-GFP (Control)

Many

Bright

Sparse

Dark

CAG-GFP (Control)

Supernova system enables sparse and bright labeling of cortical neuronsMizuno et al., Neuron (2014)

What is Supernova system and how does it work?

Vector 2 CAG Pr XFP pA

RT

STOP

RTires

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:

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

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.


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


tTARFP

3 Through binding with TRE, tTA facilitates further expression of 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

pATRE Cre

Cre




tTACAG 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

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

Axons7.5mm

Axonal boutons

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

FRTires

tTA WPRE

Flpe/FRT-based Supernova

FlpeTRE pAWPRE

CAG Pr GFP pA

rox

STOP

roxires

tTA WPRE

Dre/rox-based Supernova

DreTRE pAWPRE

These Supernova vectors can be used along with Cre.

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, thendissected 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.

The sparseness of Supernova-labeling is adjustable

0

25

50

75

100

0 1 2 3 45 50 500

(%)

No

. G

FP

+/ N

o. R

FP

+

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.

Supernova enables co-expression of multiple genes in a single cell

CAG Pr GFP pA

FRT

STOP

FRTires

tTA WPRE

FlpeTRE pAWPRE

CAG Pr RFP pA

FRT

STOP

FRTires

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.

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)

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

Labeled cell-specific gene knockout via Cre-based Supernova


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


Wild-type Knockout (KO)

Labeled cell-specific gene knockout via Cre-based

Supernova


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


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.

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.

Labeled cell-specific a2-Chn knockout via Cre-based Supernova

100 mm

500mm

CA1

IUE Cre-SnRFP → a2-Chn floxed mouse

No

. a

2-c

him

ae

rin

+/ N

o. D

AP

I+

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.

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

FRTires

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

+

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.


a2-Chn-Low

a2-Chn-High

a2-Chn-Negative

Low


20mm

20mm

Negative

High

0

20

40

60

80

100

1 2RFP+RFP-

Cell

perc

enta

ge

(%)


• 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.


Low


20mm

20mm

Negative

High

0

20

40

60

80

100

1 2RFP+RFP-

Cell

perc

enta

ge

(%)


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

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

17bpa2-Chngenome

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.

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.

Labeled cell-specific Creb knockout by Supernova-mediated CRISPR/Cas9C

on

tro

l

P8

Sn

CR

ISP

R-C

reb

50mm

Cre-SnGFP Cre-SnGFP/CREB/DAPICREBP8

50mm

Control vector: without targeting sequence in sgRNA


• 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.

Co

ntr

ol

P8

Sn

CR

ISP

R-C

reb

50mm


50mm


• 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 Creb1were transfected, CREB expression was undetectable in almost all GFP-positive neurons.

Labeled cell-specific Creb knockout by Supernova-mediated CRISPR/Cas9


Co

ntr

ol

P8

Sn

CR

ISP

R-C

reb

50mm


50mm

0

20

40

60

80

100

1 2

No.

CR

EB

+ /

No.

GF

P+

(%)

SnCRISPR-Creb

(n=85 cells)

Control

(n=111 cells)




(12 clones) 12: Edited0: wild-type

Mutations

Wild type

PAM

Deletion: 5 patternssgRNA-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.


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).

GFP

Control GFP_RNAi

LacZ

LacZ_RNAiControl

50mm

50mm

CAG-CAT-GFP reporter mouse

Rosa-LSL-LacZ reporter mouse

GF

Pin

ten

sit

y

Control RNAi

***

LacZ

inte

nsit

y

***

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.

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

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).

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

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

AAV-SnRFP was injected into the hippocampus of α2-Chn flox/floxmice 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.

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

RTires

tTA WPRE

TRE-SSR SSR pATRE

IUE-based Supernova system

Supernova based on two gene delivery systems

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!

https://www.nig.ac.jp/labs/NeurGen/

http://snsupport.webcrow.jp/

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)

@


