prefrontal cortex

Rabies Virus

B19G2

5 Genes

RVG is required for transsynaptic spread, but not transcription or viral genome replication.

(Etessami et al., 2000, Mebatsion et al., 1996 and Wickersham et al., 2007)

Cell type specific delivery

Applications and limitations

1. sparse labeling(a few cells)

Cell type?

2. sparse/intensive labeling

a) promotor too long/ unknown promotors

b)DIO/FLEX-TVA-mCherry

+DIO/FLEX-RVG

3. Retrograde tracing

Hard to control virus spreading

EnvA-TVA()

Delivering TVA and RVG into starting cells

Different ways:

1.Electroporation

2.Virus(AAV/lenti virus)

a) Promotor

CamKIIa, Somatostatin, Ef1a...

b)CRE animal

DUAL VECTORS SYSTEM

3. Transgenic animal

CRE>>TVA+RVG

Problems with

DUAL VECTORS SYSTEM

Starter cell:

TVA-mCherry+, RVG+, Rabies GFP+

TVA-mCherry+, RVG-, Rabies GFP+

=> Overestimate the number of starting populations

=> Not possible to do local input dissection(e.g., between layers in the cortex...)

Solutions

1. Tag RVG

=>sensitive to tags (personal contact with Ed Callaway)

2. All in one vector

EF1a-DIO-TVA-mCherry-t2A-RVG

=>too large 5-6 kilo basepairs

3.EF1a-DIO-TVA-t2A-RVG

=>Need to stain for different antibodies to see starter cells(e.g., unstable proteins like PV)

4.SINGLE VECTOR rabies system

EF1a-DIO-TVA-V5-t2A-RVG

SINGLE VECTOR rabies system

V5
Paramyxovirus SV5
a small epitope present on the P and V proteins of SV5
14 aa=>42bp
works good in AAV vectors
Fusion protein with TVA on C-terminal=>membrane bound

double virus system

Rabies Virus

ChR2-functional study
other opsins
other modulators
disadvantage
limited time window

3'-N-P-M-GFP-L-5'

3'-N-P-M-ChR2-mCherry-L-5'

3'-N-P-M-SSFO-EYFP-L-5'

3'-N-P-M-Jaws-GFP-L-5'