Floxopatch (also called Optopatch) mice are a sensitive, fast optogenetic tool for targeted, simultaneous optical perturbation and measurement of membrane voltage. Animals express variants of a near-infrared archaerhodopsin-based voltage indicator (QuasAr2-dark mOrange2; QuasAr2 is near infrared fluorescent, dark mOrange2 is non-fluorescent) and a blue light-gated channelrhodopsin actuator (CheRiff-EGFP; green fluorescent) in the cell plasma membrane after cre-mediated excision of a floxed Stop cassette.
Adam E. Cohen, Harvard University
Floxopatch (also called Ai130, or Optopatch) is a cre-dependent mouse line that incorporates the Optopatch2 (optogenetic) construct which enables simultaneous optical perturbation and optical readout of membrane potential. The animals express a blue-shifted channelrhodopsin actuator (CheRiff-EGFP) and a near infrared Archaerhodopsin-derived voltage indicator (QuasAr2-dark mOrange2) via a targeted floxed-Stop knock-in of the Gt(ROSA)26Sor gene.
Neurons from Floxopatch mice crossed with a variety of cre driver lines report spontaneous and optically-evoked activity in vitro, in acute brain slice, and in vivo in somatosensory ganglia.
QuasAr2 and CherRiff show greatly improved performance for all-optical electrophysiology. Neuronal excitation can be probed across spatial and temporal scales - from single dendritic spines to fields containing dozens of neurons measured in parallel, and from microsecond delays associated with action potential propagation to days-long changes in excitability.
The CheRiff construct shows good expression and membrane trafficking in cultured rat hippocampal neurons. Under typical neural culture conditions, CheRiff passes a photocurrent of 1 nA at a whole-cell illumination intensity of 22+/-10 Mw/cm2 (9-fold lower than is required for channelrhodopsin 2 (ChR2) H134R). CheRiff shows twofold larger maximal photocurrents than ChR2 H134E or ChIEF. CheRiff has an opening rate twofold faster than that of ChR2 H134R and fourfold faster than that of ChIEF. CheRiff has a similar closing rate to that of ChIEF and is 1.5-fold faster than ChR2 H134R.
Floxopatch mice were crossed with central nervous system (CNS)-specific cre driver lines, including parvalbumin-cre (Stock No. 008069), somatostatin-IRES-Cre (Stock No. 013044), and CAG-CreEr (Stock No. 004682). Histology shows patterns of EGFP fluorescence matching expression patterns reported in the Allen Brain Atlas. Examination at cellular resolution reveals that each cre line drives expression in cells with distinct locations and morphology. Expression in the soma membrane and throughout the dendritic arbor is demonstrated.
The Optopatch2 construct is safe to express in neuronal sub-populations, but not at a high level throughout the whole animal. Mice derived from crosses of the Floxopatch mice with CAG-CreEr (Stock No. 004682) and induced with tamoxifen grow more slowly after induction, have smaller body and organ sizes, and typically die between 7 and 9 days after induction. Tissues from these mice show intense EGFP fluorescence throughout the body. Littermates carrying only CAG-CreER (and not Octopatch2) show no ill-effects following tamoxifen injection.
Parvalbumin-cre (Stock No. 008069) was also used to test expression in the peripheral nervous system (PNS). Near predicted levels of expression were observed in dorsal root ganglia (DRG) neuron subtypes.
Animals homozygous for the Floxopatch allele and heterozygous for the Nav1.8-cre (Scn10a-cre) allele show a blue light threshold for triggering spikes that are 36% lower than that of mice heterozygous for both Floxopatch and Nav1.8-cre. The signal-to-noise ratio of spikes in QuasAr2 fluorescence is approximately twice as high in homozyous Floxopatch/heterozygous Nav1.8-cre mice.
To create the Floxopatch mouse, a cassette containing pCAG-FRT-loxp-Stop codons-3x SV40 polyA-loxP-Optopatch2-WPRE (woodchuck hepatitis post-transcriptional regulatory element)-bGHpA (bovine growth hormone polyadenylation signal)-AttB-pPGK-FRT-neo-pA-AttP was introduced to intron 1 of the Gt(ROSA26)Sor gene via homologous recombination in G4 129S6B6F1-derived embryonic stem cells. The donating lab reported that the resultant chimeric mice were backcrossed to C57BL/6NCrl for 6-7 generations (see SNP note below).
Optopatch2 is bicistronic construct that coexpresses QuasAr2-dark mOrange2 (an enhanced archaerhodopsin-3 (Arch)-based voltage indicator) and CheRiff-EGFP (a sensitive blue-shifted optogenetic actuator) separated by the porcine teschovirus-1 (P2A) peptide. This peptide causes a ribosomal skip, leading to near-balanced co-expression of the QuasAr2-dark mOrange2 and CheRiff-EGFP upon cre-mediated excision of the floxed Stop cassette.
To generate QuasAr2-dark mOrange2: A mutant library of Escherichia coli's archaerhodopsin-3 (Arch) D95N mutant was made using error-prone PCR. Variants were then joined with the gene encoding non-fluorescent dark mOrange2 (Y71A mutant to preserve the green/yellow portion of the spectrum for other uses). Brightness screening resulted in the identification of an enhanced variant containing five additional point mutations (P60S, T80S, D106H, F161V and H95Q, plus the original D95H). Addition of an endoplasmic reticulum export motif (ER2) from Kir2.1/Kcnj2 (FCYENE) and a trafficking sequence (a C-terminal TLM mutation) improved targeting to the plasma membrane. The resultant non-pumping variant or Arch was named QuasAr2 ("quality superior to Arch, 2").
To generate CheRiff-EGFP: A novel optogenetic actuator with a sensitive blue action spectrum (peak excitation wavelength λmax=474 nm) was identified in a screening of plant genomes and fused to EGFP fluorescent protein. EGFP expression in maintained to facilitate identification of expressing cells. Scherffelia dubia channelrhodopsin (sdChR) was derived from freshwater green algae first isolated from a small pond in Essex, England. Addition of the ER2 trafficking sequence improved membrane targeting. Introduction of an E154A mutation increased the kinetics and shifted the peak of action spectrum to λmax = 460 nm, decreasing spurious channelrhodopsin activation by red light.
A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Five of the 27 markers throughout the genome were segregating with 129, suggesting an incomplete backcross. Also, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.
|Expressed Gene||GFP, Green Fluorescent Protein,|
|Expressed Gene||RFP, Red Fluorescent Protein, coral|
|Site of Expression|
|Allele Name||targeted mutation 1, Adam Cohen|
|Allele Type||Targeted (Conditional ready (e.g. floxed), Reporter)|
|Allele Synonym(s)||Ai130; Floxopatch|
|Gene Symbol and Name||Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano|
|Gene Synonym(s)||AV258896; Gtrgeo26; Gtrgeo26; Gtrosa26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1; beta geo; expressed sequence AV258896; gene trap ROSA 26; gene trap ROSA b-geo 26|
|Expressed Gene||GFP, Green Fluorescent Protein,|
|Expressed Gene||RFP, Red Fluorescent Protein, coral|
|Strain of Origin||(129S6/SvEvTac x C57BL/6NCrl)F1|
|Molecular Note||A cassette containing pCAG-FRT-loxp-Stop codons-3x SV40 polyA-loxP-Optopatch2-WPRE (woodchuck hepatitis post-transcriptional regulatory element)-bGHpA (bovine growth hormone polyadenylation signal)-AttB-pPGK-FRT-neo-pA-AttP was introduced to intron 1. Optopatch2 is bicistronic construct that coexpresses QuasAr2-dark mOrange2 (an enhanced archaerhodopsin-3 (Arch)-based voltage indicator) and CheRiff-EGFP (a sensitive blue-shifted optogenetic actuator) separated by the porcine teschovirus-1 (P2A) peptide. This peptide causes a ribosomal skip, leading to near-balanced co-expression of the QuasAr2-dark mOrange2 and CheRiff-EGFP upon cre-mediated excision of the floxed Stop cassette.|
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