Journal: Applied and Environmental Microbiology

Article Title: Host Range of the Conjugative Transfer System of IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Characterization of Its oriT Region and Relaxase

doi: 10.1128/AEM.02359-16

Figure Lengend Snippet: Deletion derivatives of the oriTN region and their mobilization. The numerals at both ends of each fragment are the nucleotide positions in the 430-bp oriTN region. Four predicted IRs with hairpin loop structures (see Fig. 1c) are indicated by different colored boxes, DRs are indicated by arrows, and the putative IHF-binding site is depicted as a red box. The frequencies of mobilization of pNIT101 to pNIT114 from G7(NAH7K3) to KT2400Gm are expressed by the numbers of the Tcr transconjugants per donor cell. Each frequency is the mean value obtained from at least three independent experiments. Statistical analysis was performed using the t test: statistical significance (P < 0.05) in comparison with pNIT101 (*) and with pNIT104 (**).

Article Snippet: These results show that the NAH7 conjugation system has a broader host range than its replication system. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Recipient strain Mobilization frequency b pNIT6012 pNIT101 P. putida KT2440::Gm <1.1 × 10 −8 3.6 × 10 −3 P. fluorescens Pf-5G <4.0 × 10 −8 4.8 × 10 −4 P. aeruginosa KGG <3.5 × 10 −8 1.4 × 10 −4 B. multivorans ATCC 17616 <3.5 × 10 −8 5.8 × 10 −4 B. vietnamiensis G4::Gm <3.6 × 10 −8 1.5 × 10 −4 B. cenocepacia J2315::Gm <3.6 × 10 −8 7.6 × 10 −5 R. solanacearum RS1085::Gm <3.8 × 10 −8 3.8 × 10 −6 Sphingobium japonicum UT26::Gm <2.9 × 10 −8 1.0 × 10 −6 Sphingobium sp. MI1205::Gm <3.3 × 10 −8 2.4 × 10 −5 Sphingobium sp. TKS::Gm <4.3 × 10 −9 <4.4 × 10 −9 Sinorhizobium meliloti 1021 <5.6 × 10 −8 1.4 × 10 −3 Open in a separate window a Mobilization of pNIT6012 and pNIT101 from P. putida G7(NAH7K3) to the recipient strain was investigated by selecting the Tc r Gm r or Tc r Sm r transconjugants. b Mobilization frequency is expressed by the number of transconjugants per donor cell, and the mean value was obtained from at least three independent experiments.

Techniques: Binding Assay, Comparison

Journal: Applied and Environmental Microbiology

Article Title: Host Range of the Conjugative Transfer System of IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Characterization of Its oriT Region and Relaxase

doi: 10.1128/AEM.02359-16

Figure Lengend Snippet: Conjugative transfer and mobilization of plasmids a

Article Snippet: These results show that the NAH7 conjugation system has a broader host range than its replication system. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Recipient strain Mobilization frequency b pNIT6012 pNIT101 P. putida KT2440::Gm <1.1 × 10 −8 3.6 × 10 −3 P. fluorescens Pf-5G <4.0 × 10 −8 4.8 × 10 −4 P. aeruginosa KGG <3.5 × 10 −8 1.4 × 10 −4 B. multivorans ATCC 17616 <3.5 × 10 −8 5.8 × 10 −4 B. vietnamiensis G4::Gm <3.6 × 10 −8 1.5 × 10 −4 B. cenocepacia J2315::Gm <3.6 × 10 −8 7.6 × 10 −5 R. solanacearum RS1085::Gm <3.8 × 10 −8 3.8 × 10 −6 Sphingobium japonicum UT26::Gm <2.9 × 10 −8 1.0 × 10 −6 Sphingobium sp. MI1205::Gm <3.3 × 10 −8 2.4 × 10 −5 Sphingobium sp. TKS::Gm <4.3 × 10 −9 <4.4 × 10 −9 Sinorhizobium meliloti 1021 <5.6 × 10 −8 1.4 × 10 −3 Open in a separate window a Mobilization of pNIT6012 and pNIT101 from P. putida G7(NAH7K3) to the recipient strain was investigated by selecting the Tc r Gm r or Tc r Sm r transconjugants. b Mobilization frequency is expressed by the number of transconjugants per donor cell, and the mean value was obtained from at least three independent experiments.

Techniques: Plasmid Preparation, Clone Assay

Journal: Applied and Environmental Microbiology

Article Title: Host Range of the Conjugative Transfer System of IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Characterization of Its oriT Region and Relaxase

doi: 10.1128/AEM.02359-16

Figure Lengend Snippet: Mobilization of oriT N -containing plasmid to various bacterial strains a

Article Snippet: These results show that the NAH7 conjugation system has a broader host range than its replication system. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Recipient strain Mobilization frequency b pNIT6012 pNIT101 P. putida KT2440::Gm <1.1 × 10 −8 3.6 × 10 −3 P. fluorescens Pf-5G <4.0 × 10 −8 4.8 × 10 −4 P. aeruginosa KGG <3.5 × 10 −8 1.4 × 10 −4 B. multivorans ATCC 17616 <3.5 × 10 −8 5.8 × 10 −4 B. vietnamiensis G4::Gm <3.6 × 10 −8 1.5 × 10 −4 B. cenocepacia J2315::Gm <3.6 × 10 −8 7.6 × 10 −5 R. solanacearum RS1085::Gm <3.8 × 10 −8 3.8 × 10 −6 Sphingobium japonicum UT26::Gm <2.9 × 10 −8 1.0 × 10 −6 Sphingobium sp. MI1205::Gm <3.3 × 10 −8 2.4 × 10 −5 Sphingobium sp. TKS::Gm <4.3 × 10 −9 <4.4 × 10 −9 Sinorhizobium meliloti 1021 <5.6 × 10 −8 1.4 × 10 −3 Open in a separate window a Mobilization of pNIT6012 and pNIT101 from P. putida G7(NAH7K3) to the recipient strain was investigated by selecting the Tc r Gm r or Tc r Sm r transconjugants. b Mobilization frequency is expressed by the number of transconjugants per donor cell, and the mean value was obtained from at least three independent experiments.

Techniques: Plasmid Preparation

Journal: Applied and Environmental Microbiology

Article Title: Host Range of the Conjugative Transfer System of IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Characterization of Its oriT Region and Relaxase

doi: 10.1128/AEM.02359-16

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: These results show that the NAH7 conjugation system has a broader host range than its replication system. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Recipient strain Mobilization frequency b pNIT6012 pNIT101 P. putida KT2440::Gm <1.1 × 10 −8 3.6 × 10 −3 P. fluorescens Pf-5G <4.0 × 10 −8 4.8 × 10 −4 P. aeruginosa KGG <3.5 × 10 −8 1.4 × 10 −4 B. multivorans ATCC 17616 <3.5 × 10 −8 5.8 × 10 −4 B. vietnamiensis G4::Gm <3.6 × 10 −8 1.5 × 10 −4 B. cenocepacia J2315::Gm <3.6 × 10 −8 7.6 × 10 −5 R. solanacearum RS1085::Gm <3.8 × 10 −8 3.8 × 10 −6 Sphingobium japonicum UT26::Gm <2.9 × 10 −8 1.0 × 10 −6 Sphingobium sp. MI1205::Gm <3.3 × 10 −8 2.4 × 10 −5 Sphingobium sp. TKS::Gm <4.3 × 10 −9 <4.4 × 10 −9 Sinorhizobium meliloti 1021 <5.6 × 10 −8 1.4 × 10 −3 Open in a separate window a Mobilization of pNIT6012 and pNIT101 from P. putida G7(NAH7K3) to the recipient strain was investigated by selecting the Tc r Gm r or Tc r Sm r transconjugants. b Mobilization frequency is expressed by the number of transconjugants per donor cell, and the mean value was obtained from at least three independent experiments.

Techniques: Plasmid Preparation, Over Expression

Journal: Applied and Environmental Microbiology

Article Title: Host Range of the Conjugative Transfer System of IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Characterization of Its oriT Region and Relaxase

doi: 10.1128/AEM.02359-16

Figure Lengend Snippet: Primers used in this study

Article Snippet: These results show that the NAH7 conjugation system has a broader host range than its replication system. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Recipient strain Mobilization frequency b pNIT6012 pNIT101 P. putida KT2440::Gm <1.1 × 10 −8 3.6 × 10 −3 P. fluorescens Pf-5G <4.0 × 10 −8 4.8 × 10 −4 P. aeruginosa KGG <3.5 × 10 −8 1.4 × 10 −4 B. multivorans ATCC 17616 <3.5 × 10 −8 5.8 × 10 −4 B. vietnamiensis G4::Gm <3.6 × 10 −8 1.5 × 10 −4 B. cenocepacia J2315::Gm <3.6 × 10 −8 7.6 × 10 −5 R. solanacearum RS1085::Gm <3.8 × 10 −8 3.8 × 10 −6 Sphingobium japonicum UT26::Gm <2.9 × 10 −8 1.0 × 10 −6 Sphingobium sp. MI1205::Gm <3.3 × 10 −8 2.4 × 10 −5 Sphingobium sp. TKS::Gm <4.3 × 10 −9 <4.4 × 10 −9 Sinorhizobium meliloti 1021 <5.6 × 10 −8 1.4 × 10 −3 Open in a separate window a Mobilization of pNIT6012 and pNIT101 from P. putida G7(NAH7K3) to the recipient strain was investigated by selecting the Tc r Gm r or Tc r Sm r transconjugants. b Mobilization frequency is expressed by the number of transconjugants per donor cell, and the mean value was obtained from at least three independent experiments.

Techniques: Sequencing, Cloning, Amplification

Journal: Journal of Anatomy

Article Title: Scar-free cutaneous wound healing in the leopard gecko, Eublepharis macularius

doi: 10.1111/joa.12368

Figure Lengend Snippet: Summary table of the optimised immunohistochemistry and immunofluorescence protocols for proteins of interest

Article Snippet: Slides were then coverslipped using Cytoseal (TM) (Fischer Scientific). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Type Retrieval Block Primary Secondary HRP DAB WE6 IHC 12 min citrate buffer 3% NGS 1.5 h RT Mouse anti-WE6 1 : 5 (Developmental Studies Hybridoma Bank WE6-s) Biotinylated goat anti-mouse 1 : 200 (Vector Laboratories BA-9200) 1 : 200 40 s PCNA IHC None 3% NGS 1 h RT Rabbit anti-PCNA 1 : 500 (Santa Cruz Biotechnology, Inc. sc-7907) Biotinylated goat anti-rabbit 1 : 200 (Jackson ImmunoResearch Laboratories, 111-066-003) 1 : 200 25 s VEGF IHC 12 min citrate buffer 3% NGS 1 h RT Rabbit anti-VEGF 1 : 100 (Santa Cruz Biotechnology, Inc. sc-152) Biotinylated goat anti-rabbit 1 : 500 (Jackson ImmunoResearch Laboratories, 111-066-003) 1 : 200 25 s TSP-1 IHC 12 min citrate buffer 3% NGS 1 h RT Mouse anti-Thrombospondin 1 1 : 50 (Santa Cruz Biotechnology, sc-59887) Biotinylated goat anti-mouse 1 : 500 (Vector Laboratories BA-9200) 1 : 200 40 s vWF IF None 3% NGS 1 h RT Rabbit anti-vonWillebrand Factor 1 : 500 (Dako Canada, A0082) Cy3 goat anti-rabbit 1 : 400 (Jackson ImmunoResearch Laboratories, 111-165-144) α-SMA IF None 3% NGS 1 h RT Mouse anti-α-Actin 1 : 400 (Santa Cruz Biotechnology, sc-32251) Goat anti-mouse AlexaFluor-488 1 : 400 (Life Technologies, A-11001) Open in a separate window Summary table of the optimised immunohistochemistry and immunofluorescence protocols for proteins of interest Immunofluorescence To visualise blood vessels, double-labelled immunofluorescence, for both vonWillebrand factor (vWF; expressed by endothelial cells) and α-smooth muscle actin (α-SMA; expressed by perivascular cells), was performed.

Techniques: Immunohistochemistry, Immunofluorescence, Blocking Assay, Plasmid Preparation, Immunohistochemistry-IF

Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Crystal structure of the aromatic-amino-acid aminotransferase from Streptococcus mutans

doi: 10.1107/S2053230X18018472

Figure Lengend Snippet: Macromolecule-production information

Article Snippet: Macromolecule-production information is summarized in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Source organism S. mutans DNA source S. mutans strain UA159 (ATCC 700610) Forward primer 5′-CGC GGATCC ATGGATTTGAGTAAACGTTTTA-3′ Reverse primer 5′-CCG CTCGAG TTAGTCTGCATATTGCTCC-3′ Cloning vector pGEX-6P-1 Expression vector pGEX-6P-1 Expression host E. coli strain BL21 (DE3) Complete amino-acid sequence of the construct produced GPMDLSKRFNKNLNKIEVSMIRQFDQSISDIPDVLKLTLGEPDFATPKHIKEAAKRAIDADESHYTGMAGLLALRQAASAFVKEKYHLTYNPDNEILVTIGATEALSASLTAILEPGDKVLLPAPAYPGYEPVVNLVGAEVVEIDTRSNDFVLTPEMLEEAILKEGEALKAVILNYPTNPTGVTYSRQQIKNLAEVLKKYPIFVISDEVYAELTYTGESHVSIAEYLPDQTILISGLSKSHAMTGWRLGLIFAPAVLTAQLIKSHQYLVTAATTSVQFAAIEALTNGKDDALPMKEEYIKRRDYIIEKMEAMKFKIIKPDGAFYIFAKIPVAQGQDSFKFLQDFAKEKAVAFIPGVAFGKYGEGYLRISYAASMETIKEAMKRLKEFMEQYAD Open in a separate window caption a8 Macromolecule-production information 2.2.

Techniques: Cloning, Plasmid Preparation, Expressing, Sequencing, Construct, Produced

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 3. Generation of Tg-MPP1 mice. A, Upper panel: scheme of the plasmid used for the generation of Tg-MPP1 mice. Lower panel: PCR genotyping of ear- punch biopsies from 11 Tg-MPP1-positive mice with stable integration of the transgenic MPP1 cDNA into the genomic DNA. The negative control (-) did not contain genomic DNA, and the linearized MPP1 plasmid DNA (P) was used as a positive control. The lane marked with M, is the DNA marker. B, Immunoblot detection of the MPP1 protein in heart protein extracts from Tg-MPP1 mice and non-transgenic B6 mice. The left panel is a representative immunoblot, and the right panel shows quantitative data (mean ± s.d., n = 4 mice per group). The p- value is indicated and was determined by the unpaired, two-tailed, t-test. The lower panel is a control immunoblot detecting α-tubulin. C, As a specificity control of the monoclonal anti-MPP1 antibody, immunoblot detection of MPP1 in MPP1-transfected HEK cells was performed in comparison to mock- transfected HEK cells. The lower blot shows a loading control detecting GAPDH.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Plasmid Preparation, Transgenic Assay, Negative Control, Positive Control, Marker, Western Blot, Two Tailed Test, Control, Transfection, Comparison

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 2. Upregulation of the MAGUK family protein, MPP1, in three different heart failure models. A,B, Probe set intensities of cardiac Mpp iso forms were determined by whole genome microarray gene expression profiling of the AAC-induced heart failure model in comparison to sham-operated con trols (A), and of Apoe−/− mice with long-term atherosclerosis-induced heart failure in comparison to age-matched non-transgenic B6 mice (B). Affymetrix IDs of probe sets detecting Mpp1, Mpp2, Mpp3, Mpp4, Mpp5, Mpp6, and Mpp7 are indicated. Data are mean values ± s.d. (four hearts per microarray chip with two microarray chips per group). Probe set intensities are taken from NCBI GEO dataset GSE25765. C, Cardiac transcript levels of Mpp isoforms in 8-month-old, male Tg-RKIP mice were determined by NGS in comparison to age- and sex- matched, non-transgenic FVB controls (NCBI GEO dataset GSE191316) (mean ± s.d., n = 3 mice per group). Statistically significant differences between transcript levels of the heart failure groups and the respective control group were determined by Tukey’s test, and are indicated for each individual MAGUK gene (A,B,C). P-values for statistically different MAGUK genes are indicated. All other MAGUK genes were not significantly different (n.s.) between the heart failure and control groups.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Microarray, Gene Expression, Comparison, Transgenic Assay, Control

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 4. Tg-MPP1 mice develop features of heart failure with cardiac enlarge ment at an age of 8 months. A, Echo cardiographic measurement of the left ventricular ejection fraction (LVEF, %), the fractional shortening (FS, %), the left ventricular internal diameter in diastole (LVIDd), and the left ventricular internal diameter in systole (LVIDs) of 8-month- old, male Tg-MPP1 mice, and sex- and age-matched, non-transgenic B6 mice. Echocardiographic measurements were performed under anesthesia. B, Determi nation of the body weights (BW), heart weights (HW), and the heart weight to body weight ratios (HW/BW) of 8-month- old, male Tg-MPP1 mice, and of sex- and age-matched, non-transgenic B6 mice. Data (A,B) are the mean ± s.d., n = 6 mice per group. P-values were determined by the unpaired, two-tailed t-test. C, Immu nohistological detection of MPP1 on heart sections of Tg-MPP1 mice in comparison to those of non-transgenic B6 mice (n = 4 mice/group; bar: 2 mm). Sections were stained with the anti-MPP1 antibody (MPP1) and counterstained with hema toxylin (HE). The right panels show higher magnification images of representative sections from a Tg-MPP1 mouse and a non- transgenic B6 control (bar: 20 μm).

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Transgenic Assay, Two Tailed Test, Comparison, Staining, Control

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 5. Co-localization of AGTR1 with MPP1 in vivo, and increased cardiac AGTR1 protein levels in Tg- MPP1 mice. A, Immunofluorescence detection of MPP1 and AGTR1 on cardiac cryosections from Tg-CMV- AGTR1-Cerulean mice shows co-localization of AGTR1 with MPP1 on sarcolemmal membranes (yellow). MPP1 was stained with mouse monoclonal anti-MPP1 antibody (red), AGTR1-Cerulean was stained with rabbit poly clonal anti-GFP antibodies (green), and nuclei were stained with DAPI (blue). The immunofluorescence co- localization study shows cryosections from four different mice (bar: 40 μm). B, Cardiac AGTR1-specific binding sites were determined on sarcolemmal mem branes of Tg-MPP1 mice and non-transgenic B6 mice by radioligand binding with Sar1,[125I]Tyr4,Ile8-angiotensin II. Data are shown as mean values ± s.d., n = 6 mice per group. The p-value was determined by the unpaired, two- tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: In Vivo, Immunofluorescence, Staining, Binding Assay, Transgenic Assay, Two Tailed Test

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 6. MPP1 increased the cellular contents of AGTR1eYFP in HEK cells. A,B, Cellular AGTR1eYFP levels were increased by co-transfection of HEK293 cells with an MPP1-encoding pcDNA3 expression plasmid (+). Control cells were transfected with the pcDNA3 plasmid without insert (-). Panel (A) shows cellular AGTR1eYFP fluorescence peak intensities at an emission wavelength of 527 nm, and panel (B) shows representative AGTR1eYFP fluorescence emis sion spectra without (grey) and with MPP1-encoding plasmid co-transfection (red). The black line shows a spectrum of control cells transfected with pcDNA3 without insert (Cont.). C,D, Co-transfection of the MPP1-encoding plasmid did not significantly alter cellular ADRB1eYFP levels. Control cells were trans fected with the pcDNA3 plasmid without insert (-). Panel (C) shows cellular ADRB1eYFP fluorescence peak intensities at an emission wavelength of 527 nm, and panel (D) shows representative fluorescence emission spectra of ADRB1eYFP-expressing cells without and with MPP1-encoding plasmid co- transfection. Data (A,C) show mean values ± s.d. (n = 8 biological replicates). P-values were determined by Tukey’s test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Cotransfection, Expressing, Plasmid Preparation, Control, Transfection, Fluorescence

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 7. AGTR1-(1–319)-eYFP with deletion of the carboxyl terminal tail is also enhanced by MPP1 in HEK cells. A, Cellular fluorescence peak intensities at an emission wavelength of 527 nm were deter mined of HEK cells with expression of the full-length AGTR1-(1–359)-eYFP without (-) and with (+) co- transfection of the MPP1-encoding plasmid, and of HEK cells with expression of the truncated AGTR1- (1–319)-eYFP without (-), and with (+) co- transfection of MPP1. Data are mean values ± s.d. (n = 10 biological replicates). P-values were deter mined by Tukey’s test. B, Topological scheme of the full-length AGTR1-(1–359) protein sequence. Trun cated residues of AGTR1-(1–319) are marked in red. The AGTR1 topology was derived from Uniprot (P30556 AGTR1_Human), and the scheme was drawn with Protter, version 1.0. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Fluorescence, Expressing, Cotransfection, Plasmid Preparation, Sequencing, Derivative Assay

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 9. The AGTR1-enhancing effect mediated by MPP1 requires all functional domains of MPP1. A, Scheme of MPP1 functional domains, and of the two MPP1 fragments 1–267 and 268–466, which were tested. B, Cellular fluorescence peak intensities at an emission wavelength of 527 nm were determined of AGTR1eYFP-expressing HEK cells without (-) and with (+) co- transfection of MPP1-encoding plasmid, MPP1-(1–267)-encoding plasmid, MPP1-(268–466)-encoding plasmid, or MPP1-(1–267) and MPP1-(268–466)- encoding plasmids together. Data are presented as mean values ± s.d. (n = 4 biological replicates). P-values were determined by Tukey’s test.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Functional Assay, Fluorescence, Expressing, Cotransfection, Plasmid Preparation

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 8. Deletion of a putative internal PDZ domain-binding motif in AGTR1-(1–319)-(Δ213-220)-eYFP abolishes the AGTR1-enhancing effect by MPP1 in HEK cells. A, Topological scheme of the AGTR1-(1–359) protein sequence, in which deletions made in construct AGTR1-(1–319)-(Δ213-220) are marked in red. The scheme was drawn with Protter, version 1.0. Residues 213–220 at the beginning of the third intracellular loop of AGTR1 include the sequence “Y-T-L-I”, which could be an internal PDZ domain-binding motif, which is defined by “X-S/T-X-ϕ“ where “X” can be any amino acid, and “ϕ“ is a hydrophobic amino acid. B, Cellular fluorescence peak intensities at an emis sion wavelength of 527 nm were determined of HEK cells without (-) and with stable MPP1 (+) expression, and transfection of AGTR1-(1–319)-eYFP, or AGTR1-(1–319)-(Δ213-220)-eYFP with deletion of a putative internal PDZ domain-binding motif (Δ213-220). Data are presented as mean values ± s.d. (n = 3 biological replicates). P-values were determined by Tukey’s test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Binding Assay, Sequencing, Construct, Fluorescence, Expressing, Transfection

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 10. Upregulation of cardiac Mpp1 transcript levels by diabetes- induced cardiac dysfunction and by Hdac3 deficiency in rodents. A, Car diac Mpp1 transcript levels were up-regulated in rats with diabetes-induced cardiac dysfunction. Data were retrieved from the GEO profile GDS3153 (31), probe set ID 1389963_at of the Affymetrix Rat Expression 230A Array. Hearts were obtained from 12-week-old rats with four weeks of streptozotocin-induced diabetes and from control rats (mean ± s.d., n = 3 hearts per group). B, Upregulation of cardiac Mpp1 in hearts from 6-week-old mice with Hdac3- deficiency (Hdac3 KO) in heart and skeletal muscle (HDAC3fl/fl/MCK-Cre), which develop a severe hypertrophic cardiomyopathy on a high fat diet (32). Control hearts were isolated from wild-type mice (HDAC3fl/fl). Data were taken from the GEO profile GDS4886, probe set ID 106447481 of the Affymetrix Mouse Gene 1.0 ST Array (mean ± s.d., n = 4 male mice per group). P-values were determined by the unpaired, two-tailed t-test.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Expressing, Control, Isolation, Two Tailed Test

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 11. Detection of increased MPP1 transcript levels in peripheral blood mononuclear cells of old human research participants. A-F, Transcript levels of MPP1 (A), GRK2 (B), GRK3 (C), DUSP3 (D), LRRN3 (E), and CD27 (F) in PBMC from old (age: 75–89 years, y; n = 5) human research participants were determined by whole genome microarray gene expression profiling. PBMC isolated from middle-aged research participants (age: 35–50 years, y; n = 4) served as the control group. Data are shown as mean values ± s.d. P-values were determined by the two- tailed (A,B,D,E,F), or one-tailed (C), unpaired t-test.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Microarray, Gene Expression, Isolation, Control, Two Tailed Test, One-tailed Test

Journal: Molecular and Cellular Biology

Article Title: EWSR1-FLI1 Activation of the Cancer/Testis Antigen FATE1 Promotes Ewing Sarcoma Survival

doi: 10.1128/mcb.00138-19

Figure Lengend Snippet: Fig. 1: FATE1 is a Target of the Oncogenic Transcription Factor EWSR1-FLI1. (A) FATE1 607

Article Snippet: For shRNA experiments pLKO.1 vectors from The RNA 312 Consortium (TRC) expressing FATE1 targeted shRNAs (A7: TRCN0000130192 and A9: 313 on M ay 4, 2019 by guest http://m cb.asm .org/ D ow nloaded from 12 TRCN0000146597) were obtained from the UNC Lentiviral core and a non-targeting shRNA in pLKO.1 314 (shSCR) was used as a control (Addgene).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: EWSR1-FLI1 Activation of the Cancer/Testis Antigen FATE1 Promotes Ewing Sarcoma Survival

doi: 10.1128/mcb.00138-19

Figure Lengend Snippet: Fig. 5: FATE1 Destabilizes BNIP3L. (A, B) 72 hours after siRNA transfection, WCLs were collected 655

Article Snippet: For shRNA experiments pLKO.1 vectors from The RNA 312 Consortium (TRC) expressing FATE1 targeted shRNAs (A7: TRCN0000130192 and A9: 313 on M ay 4, 2019 by guest http://m cb.asm .org/ D ow nloaded from 12 TRCN0000146597) were obtained from the UNC Lentiviral core and a non-targeting shRNA in pLKO.1 314 (shSCR) was used as a control (Addgene).

Techniques: Transfection

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Application in descending pathways to the spinal cord. A, Retrograde labeling of spinal-projecting cortical and subcortical neuronal populations following injections of AAVretro-hSyn-Cre (red) and AAVretro-hSyn-GFP (green) into the left side of the cervical and lumbar spinal cord, respectively, in Ai14 mice. Scale bars: A–C, 500 µm. B, Lack of axonal projections from spinal cord to selected brain regions following cervical injection of AAV1-hSyn-GFP (green) and lumbar injection AAV1-CAG-tdTomato (red). C, Corresponding injections of scAAV1-hSyn-Cre (red; 100 nl injection volume) into different spinal-projecting brain regions (A, top) in Ai14 mice. D, Different patterns of transsynaptic labeling at cervical, thoracic, and lumbar segments of the spinal cord for each injection following a 2 week postinjection survival time. Each column corresponds to the injection site shown in C. Scale bars, 250 µm. E, Quantification of the average number of anterograde transsynaptically labeled cells per coronal section of cervical (C) thoracic (T), or lumbar (L) spinal cord for each injection (n = 1 mouse each). Error bar = SD.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Labeling, Injection

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Comparison of anterograde transsynaptic spread, toxicity, and retrograde transport potential. A, Injection of AAV1-hSyn-Cre-WPRE in V1 of Ai14 mice (top). Overexpression of Cre may result in cell death at the injection site, as seen by a reduction in Nissl stain intensity and irregular cell morphology (middle). Bottom, Anterograde transsynaptically labeled neurons in SC. B, Injection of scAAV1-hSyn-Cre in Ai14 mice (top). This virus lacks the WPRE enhancer and shows no apparent toxicity in neurons at the injection site (middle). Bottom, Anterograde transsynaptically labeled neurons in SC. C, AAV1-hSyn-Flp injection in Frt-GFP mice (top). This virus also lacks the WPRE enhancer and shows no apparent toxicity (middle). Bottom, Anterograde transsynaptically labeled neurons in SC. All injections 60 nl total volume, 4 week postinjection survival time. Scale bars, 500 µm. D, Quantification of total number of cells labeled in SC for different Cre or Flp-expressing viruses injected into V1 (4 week postinjection survival, 60 nl injection, n = 4 mice each). Error bar = SD. E, Injection of AAVretro-hSyn-Cre in IC of Ai14 mice (left). Retrograde labeling in A1 (middle), and close-up of dashed region (right). Scale bars: left and middle, 500 µm; right, 100 µm. F, Injection of scAAV1-hSyn-Cre in IC of Ai14 mice(left). Retrograde labeling in A1 (middle), and close-up of dashed region (right). Scale bars: left and middle, 500 µm; right, 100 µm. G, Injection of scAAV1-hSyn-Cre in A1 of Ai14 mice (left). Anterograde transsynaptic labeling of cells in IC (middle), and close up of dashed region (right). Scale bars: left and middle, 500 µm; right, 100 µm. H, Quantification of retrograde labeling in A1 following injection of scAAV1-hSyn-Cre or AAVretro-hSyn-Cre in IC (n = 3 mice for each group). All injections 80 nl total volume, 2 week postinjection survival time. Error bar = SD. ***p < 0.001, t test. I, Quantification of anterograde transsynaptic labeling in IC following injection of scAAV1-hSyn-Cre or AAVretro-hSyn-Cre in A1 (n = 3 mice for each group). All injections 80 nl total volume, 2 week postinjection survival time. Error bar = SD. *p < 0.05, t test.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Comparison, Injection, Over Expression, Staining, Labeling, Virus, Expressing

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Anatomical evidence for the synaptic specificity of viral spread. A, Illustration shows that following injection in an upstream brain region, AAV1-Cre is transported down axons and may be released through the synapse to transduce postsynaptically connected neurons (red cells, in Cre-dependent tdTomato background). The extent to which extra-synaptic release of virus may contribute to the local transduction of unconnected cell types (gray cells) remains unclear. B, Strategy for testing the synaptic specificity of viral spread in an anatomically defined circuit. Postsynaptic labeling was examined in the simple lobule of the CB following injections in the inferior olive or PN (left). Mossy fiber afferents from the PN are known to innervate GCs but not PCs (right). On the other hand, climbing fiber afferents from the IO pass through the granule layer and selectively innervate PCs, but not GCs. Locations of cell bodies in different layers of the CB are indicated by dashed lines (molecular layer, Purkinje layer, or granule layer). C, Approach for labeling neurons postsynaptic to mossy fibers in the CB. The scAAV1-hSyn-Cre was injected into the PN of Ai14 x GAD67-GFP transgenic mice. Bottom, Example injection site (red). Blue, fluorescent Nissl stain. Scale bar, 500 µm. D, A coronal section through simple lobule showing pontine afferents and postsynaptic neurons labeled in the granule layer of the cerebellum (red). GAD67-GFP+ PCs and molecular layer interneurons are labeled in green. Blue, Fluorescent Nissl stain. Scale bar, 250 µm. E, Higher-magnification view of the dashed region shown in D. TdTomato+/GFP− GCs (red; costained with Nissl, blue, arrowheads) and mossy fiber terminals (red) were observed in the granule layer, along with large GAD67-GFP+ neurons (yellow, asterisk). Scale bar, 25 µm. F, Approach for labeling neurons postsynaptic to climbing fibers in CB. The scAAV1-hSyn-Cre was injected into the IO of Ai14 x GAD67-GFP mice. Bottom, Example injection site (red). Scale bar, 500 µm. G, Climbing fiber afferents and postsynaptic neurons (red) labeled in the granule, Purkinje, and molecular layers of the simple lobule. Most labeled neurons in the granule cell layer colocalized with GAD67-GFP+ expression. Scale bar, 250 µm. Solid box shows close up of a MLI. Scale bar, 25 µm. H, Higher-magnification view of the dashed region shown in G. Cre+/tdTomato+/GFP+ neurons were observed in the granule layer (yellow, asterisks), Purkinje cell layer (yellow, arrowhead), and molecular layer (cells shown in G). I, Quantification of the total number of GCs (top) and PCs (bottom) counted across four sections of the simple lobule for each animal injected in PN or IO (mean plotted for n = 4 animals for each pathway). Error bar = SD. ***p < 0.001, t test.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Injection, Transduction, Virus, Labeling, Transgenic Assay, Staining, Expressing

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Tetanus toxin inhibition of viral spread. A, Experimental design and timeline of virus injections. AAV trafficked to the synapse may be released through synaptic vesicles in a VAMP2-dependent manner (top). Tetanus toxin cleaves VAMP2, preventing synaptic vesicle fusion and potential release of AAV. B, Control experiment. AAV1-hSyn-GFP injection in V1 followed by scAAV1-hSyn-Cre injection 2 weeks later in Ai14 mice (top). 2 weeks after the second injection, GFP+ axons (green) and anterograde transsynaptically labeled neurons (red cells) were observed in SC (middle, 10×; bottom, 40×). Blue, fluorescent Nissl stain. C, AAVDJ-CMV-TeNT-P2A-GFP injection in V1 followed by scAAV1-hSyn-Cre injection 2 weeks later in Ai14 mice (top). 2 weeks after the second injection, GFP+/TeNT+ axons (green) were found in SC, however, very few transsynaptically labeled neurons (red cells) were observed. Scale bars: B, C, top, 500 µm; middle, 250 µm; bottom, 25 µm. D, Quantification of number of anterograde transsynaptically labeled cells in SC for each injection time point (n = 8 mice for control and 14 d groups, n = 4 mice for the remaining groups). Error bar = SD. ***p < 0.001; n.s., no significance, t test.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Inhibition, Virus, Control, Injection, Labeling, Staining

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Forward mapping of topographically organized brain regions. A, Schematic diagram depicting the parcellation of brain regions (in Y) based on their topographical input (from X) using an anterograde transsynaptic approach. Secondary injections of AAV1-Flex-GFP enable further mapping of axonal outputs (to Z) for any given input-defined subregion. A retrograde approach may not provide access to the same population because of collateralization of divergent outputs. B, Top, tdTomato expression at the injection site, MOp-ul, following first injection of scAAV1-hSyn-Cre. Bottom, Cre+/GFP+ neurons are shown in PN following secondary injection of AAV1-hSyn-Flex-GFP in PN. Scale bar, 250 µm. C, Axonal projections to the cerebellum from PN neurons defined by their input from MOp-ul. Axons primarily target the contralateral parafloccular sulcus (PflS; top left) and the ventral PRM. Scale bar, 500 µm. D, Top, first injection in MOp-ll. Bottom, Cre+/GFP+ neurons in PN following secondary injection of AAV1-hSyn-Flex-GFP in PN. Scale bar, 250 µm. E, Axonal targeting in CB. Output was primarily restricted to the (COP (top right and bottom), but also collateralize more sparsely to lobules III, IV, and V (top left). Scale bars, 500 µm. F, Quantification of the density of axon terminals in PRM or COP for PN neurons defined by their input from MOp-ul (blue) or MOp-ll (green; n = 3 mice for each group). Values represent the fraction of fluorescent axon signal divided by the total area of the target lobule. Error bar = SD. ***p < 0.001, t test. G, Schematic summary of axonal projections to cerebellum from MOp-ul and MOp-ll-recipient PN neurons (green and blue, respectively). Posterior view of the cerebellum is shown.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Expressing, Injection

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Application with sparse labeling approaches for reconstructing single neuron morphology. A, For a given Cre+ cell population (red), sparse labeling (green) may be achieved by coinjecting AAV1-DIO-Flp at increasingly lower titers along with high titer AAVDJ-fDIO-YFP. To establish a titering curve, PV neurons in V1 were targeted with coinjections of AAVDJ-fDIO-YFP (final titer: 1.2 × 1013 GC/ml) and AAV1-DIO-Flp (final titers: 7.5 × 1010, 109, or 108 GC/ml) in PV-Cre x Ai14 mice. B, Examples of YFP cell labeling (green) achieved at each titer step. Red, PV-Cre+/tdTomato+ cells. Blue, Fluorescent Nissl. Scale bar, 250 µm. C, 40× magnification of a YFP+ PV neuron labeled in (B, middle). Scale bar, 25 µm. D, Quantification of the number of YFP+/PV+ cells labeled at each titer step (n = 4 mice each). Error bar = SD. E, Injection of scAAV1-hSyn-Cre in the ACA of GAD67-GFP x Ai14 mice transsynaptically labels neurons in PAG. Labeled cell density is greatest in the PAGdl (middle), and both inhibitory (GFP+/tdTomato+) and presumed excitatory (GFP-/tdTomato+) cell types are labeled (bottom; 40× magnification). Scale bars: middle, 250 µm; bottom, 50 µm. F, Strategy for sparse labeling of input- and genetically-defined cell populations. AAV1-DIO-Flp titer for injection in ACA is reduced to 1.5 × 1012 GC/ml to achieve sparse anterograde transsynaptic labeling in PAGdl. Individual Vglut2-Cre+/Flp+ neurons may then be targeted with an injection of high titer AAVDJ-fDIO-YFP to specifically label glutamatergic neurons in PAGdl that receive input from ACA. G) Example of a single Vglut2-Cre+/Flp+ neuron labeled in PAGdl (green). An additional neuron was found in the superior colliculus (arrowhead). Right, 40× magnification of YFP+ (green, top) and Vglut2-Cre+/Tom+ (red, bottom) neuron in PAGdl. Blue, Fluorescent Nissl. Scale bars: left, 250 µm; right, 25 µm. H, 40× magnification of local dendrites and axonal projection of the YFP+ PAGdl neuron shown in G. Scale bar, 50 µm.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Labeling, Injection

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Verification of functional synaptic connectivity. A, Strategy for slice recording from transsynaptically labeled neurons in the IC (red) following coinjection of scAAV1-hSyn-Cre and AAV1-DIO-ChR2-YFP into A1. B, ChR2-expressing axon terminals (green) surrounding a tdTomato-labeled neuron and neighboring nonlabeled neurons (blue, Nissl stain). Scale bar, 25 µm. C, Average LED-evoked excitatory (recorded at −70 mV) and inhibitory (0 mV) currents in an example tdTomato+ IC neuron before (left) and after (right) perfusing in TTX and 4-AP. LED stimulation is marked by a blue bar. D, Fraction of transsynaptically labeled (red) and neighboring nonlabeled (gray) neurons showing monosynaptic excitatory currents in response to LED stimulation. ***p < 0.001, χ2 test, 28 cells in each group. E, Summary of amplitudes of average monosynaptic excitatory currents evoked by LED for all labeled (red) and nonlabeled (gray) recorded neurons (neurons showing zero currents were excluded). Error bar = SD. There is n.s., no significance, unpaired t test.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Functional Assay, Labeling, Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: List of viruses used in this study

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Plasmid Preparation, Virus

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Accessing topographically precise, input-defined cell populations through corticofugal pathways. A, Strategy for labeling cell populations that receive input from upper limb- (green) and lower limb- (red) related primary motor cortex in Ai14 x Frt-GFP (Cre and Flp reporter) mice. B, Injection sites for AAV1-hSyn-Flp in MOp-ul (top, green) and scAAV1-hSyn-Cre in MOp-ll (bottom, red) in an Ai14 x Frt-GFP mouse. Scale bar, 500 µm. C, Anterograde transsynaptic labeling of cells that receive input from upper limb- (green) or lower limb- (red) related primary motor cortex. Many closely apposed, non-overlapping cell populations were observed in mid- and hindbrain structures, including the PN (second row, right). None of the structures shown project back to motor cortex, with the exception of the thalamus (top row, third from left), which may contain both retrograde and anterograde transsynaptic labeling of cell bodies. Scale bars, 250 µm. GR, Gracile nucleus; MA3, medial accessory oculomotor nucleus; MARN, magnocellular reticular nucleus; PRN, pontine reticular nucleus.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Labeling, Injection