Fig. 1
H129-dgK-G4: construction, production, and illustration of anterograde monosynaptic tracing. A. Schematic genome structure of H129-dgK-G4. Based on the previously created H129-G4, H129-dgK-G4 was generated by knocking out the gK gene and replacing it with the ampicillin resistance gene (AmpR). B. Reconstitution and propagation of H129-dgK-G4. The BACH129-dgK-G4 DNA was transfected into Vero cells stably expressing wildtype gK (indicated with the orange triangle) (Vero-gKwt) to reconstitute the recombinant virus with wildtype gK residing on the viral envelope, designated as H129-dgK-G4(gKwt) (left panel). As the seed virus, H129-dgK-G4(gKwt) was further propagated in Vero cells stably expressing gKmut (Vero-gKmut). The obtained recombinant virus is pseudotyped with the mutant gK (gKmut, indicated with the orange circle), and designated as H129-dgK-G4(gKmut) (right panel). To simplify the labeling, H129-dgK-G4 represents the H129-dgK-G4(gKmut), unless specified. C. Schematic genome structure of the AAV helpers for H129-dgK-G4 monosynaptic tracing. AAV2/9 expressing mCherry and gKwt is used as the helper virus for monosynaptic tracing. To simplify the labeling, the gK in AAVs all indicates gKwt. AAV2/9-mCh-gK expresses gK and mCherry constitutively under the control of EF1α promoter, while AAV2/9-DIO-mCh-gK expresses them only in the presence of Cre recombinase. D. Schematic mechanism of H129-dgK-G4 monosynaptic tracing. After infecting the starter neurons, H129-dgK-G4 replicates its genome and synthesizes viral proteins as well as the fluorescent genes, and thus brightly labels the neurons. The deficiency of gK impairs viral egress and transmission (left panel). Coinfected AAV helper expresses gK in trans and assists the egress of H129-dgK-G4, which in turn anterogradely transmits to and labels the post-synaptic neurons (right panel). Then, H129-dgK-G4 is restrained in the 2nd order neurons, and does not further spread due to the lack of the AAV helper