aav dj egfp  (Vector Biolabs)

Journal: Virology Journal

Article Title: Tropism of adeno-associated virus serotypes in mouse lungs via intratracheal instillation

doi: 10.1186/s12985-024-02575-9

Figure Lengend Snippet: Analysis of infection efficiency of AAVs in the mouse lungs. The ratio of eGFP + cells to DAPI + cells. 2746, 695, 0, 115, 26, 2435, 0, 2588, 3118, 2919, 205, 1386, 2366, 1016, 1525, 1673, 1352, 2200, 1681, 2078 eGFP + cells were analyzed for AAV1, AAV2, MyoAAV 2A, AAV2-retro, AAV3b, AAV4, MyoAAV 4A, AAV5, AAV6, AAV6.2, AAV7m8, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8, AAVie, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, and 9263, 9804, 9701, 8762, 10,703, 9052, 9592, 9193, 9145, 9217, 10,157, 10,084, 10,272, 10,672, 10,330, 10,242, 9735, 10,423, 9715, 9958 DAPI + cells, respectively. n = 3

Article Snippet: AAVs serotypes (PackGene Biotech) used in this study were as follows: AAV1-CAG-Dio-eGFP-WPRE-pA, AAV2-CAG-Dio-eGFP-WPRE-pA, MyoAAV 2A-CAG-Dio-eGFP-WPRE-pA, AAV2-retro-CAG-Dio-eGFP-WPRE-pA, AAV3b-CAG-Dio-eGFP-WPRE-pA, AAV4-CAG-Dio-eGFP-WPRE-pA, MyoAAV 4A-CAG-Dio-eGFP-WPRE-pA, AAV5-CAG-Dio-eGFP-WPRE-pA, AAV6-CAG-Dio-eGFP-WPRE-pA, AAV6.2-CAG-Dio-eGFP-WPRE-pA, AAV7m8-CAG-Dio-eGFP-WPRE-pA, AAV8-CAG-Dio-eGFP-WPRE-pA, AAV9-CAG-Dio-eGFP-WPRE-pA, AAVrh10-CAG-Dio-eGFP-WPRE-pA, AAV-DJ-CAG-Dio-eGFP-WPRE-pA, AAV-DJ/8-CAG-Dio-eGFP-WPRE-pA, AAV-ie-CAG-Dio-eGFP-WPRE-pA, AAV-PHP.B-CAG-Dio-eGFP-WPRE-pA, AAV-PHP.eB-CAG-Dio-eGFP-WPRE-pA, and AAV-PHP.S-CAG-Dio-eGFP-WPRE-pA. For each mouse, 40 μL of AAV-containing solution (10 [ ] GC/mL) was injected into the lungs through the trachea.

Techniques: Infection

Journal: Virology Journal

Article Title: Tropism of adeno-associated virus serotypes in mouse lungs via intratracheal instillation

doi: 10.1186/s12985-024-02575-9

Figure Lengend Snippet: AAV8-eGFP expression in the lungs infected by AAV8 in mice. a Immunostaining for eGFP (green), SCGB1A1 (red), and DAPI staining (blue) of 2-month-old mouse lungs infected with AAV8-eGFP. b Immunostaining for eGFP (green), RFX3 (red), and DAPI staining (blue) of 2-month-old mouse lungs infected with AAV8-eGFP. c Immunostaining for eGFP (green), Acetylated-α-tubulin (red), and DAPI staining (blue) of 2-month-old mouse lungs infected with AAV8-eGFP. d Immunostaining for eGFP (green), CGRP (red), and DAPI staining (blue) of 2-month-old mouse lungs infected with AAV8-eGFP. e Immunostaining for eGFP (green), HOPX (red), and DAPI staining (blue) of 2-month-old mouse lungs infected with AAV8-eGFP. f Immunostaining for eGFP (green), SFTPC (red), and DAPI staining (blue) of 2-month-old mouse lungs infected with AAV8-eGFP. g Immunostaining for eGFP (green), α-SMA (red), and DAPI staining (blue) of 2-month-old mouse lungs infected with AAV8-eGFP. h Immunostaining for eGFP (green), ERG (red), and DAPI staining (blue) of 2-month-old mouse lungs infected with AAV8-eGFP. Scale bars: 100 μm

Article Snippet: AAVs serotypes (PackGene Biotech) used in this study were as follows: AAV1-CAG-Dio-eGFP-WPRE-pA, AAV2-CAG-Dio-eGFP-WPRE-pA, MyoAAV 2A-CAG-Dio-eGFP-WPRE-pA, AAV2-retro-CAG-Dio-eGFP-WPRE-pA, AAV3b-CAG-Dio-eGFP-WPRE-pA, AAV4-CAG-Dio-eGFP-WPRE-pA, MyoAAV 4A-CAG-Dio-eGFP-WPRE-pA, AAV5-CAG-Dio-eGFP-WPRE-pA, AAV6-CAG-Dio-eGFP-WPRE-pA, AAV6.2-CAG-Dio-eGFP-WPRE-pA, AAV7m8-CAG-Dio-eGFP-WPRE-pA, AAV8-CAG-Dio-eGFP-WPRE-pA, AAV9-CAG-Dio-eGFP-WPRE-pA, AAVrh10-CAG-Dio-eGFP-WPRE-pA, AAV-DJ-CAG-Dio-eGFP-WPRE-pA, AAV-DJ/8-CAG-Dio-eGFP-WPRE-pA, AAV-ie-CAG-Dio-eGFP-WPRE-pA, AAV-PHP.B-CAG-Dio-eGFP-WPRE-pA, AAV-PHP.eB-CAG-Dio-eGFP-WPRE-pA, and AAV-PHP.S-CAG-Dio-eGFP-WPRE-pA. For each mouse, 40 μL of AAV-containing solution (10 [ ] GC/mL) was injected into the lungs through the trachea.

Techniques: Expressing, Infection, Immunostaining, Staining

Journal: Virology Journal

Article Title: Tropism of adeno-associated virus serotypes in mouse lungs via intratracheal instillation

doi: 10.1186/s12985-024-02575-9

Figure Lengend Snippet: Quantification of pulmonary epithelial cells that are infected by AAVs. a Ratio of SCGB1A1 + cells that are eGFP + . 1754, 260, 0, 0, 94, 1308, 0, 1282, 1240, 1470, 74, 126, 0, 0, 126, 254, 0, 276, 366, 0 for eGFP + SCGB1A1 + cells were analyzed for AAV1, AAV2, MyoAAV 2A, AAV2-retro, AAV3b, AAV4, MyoAAV 4A, AAV5, AAV6, AAV6.2, AAV7m8, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8, AAVie, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, and 2456, 3116, 2478, 2856, 2094, 1904, 2226, 1904, 1586, 1932, 1724, 1940, 1204, 1372, 2824, 2996, 1778, 2300, 2460, 1736 for SCGB1A1 + cells, respectively. n = 3. b Ratio of RFX3 + cells that are eGFP + . 612, 0, 0, 0, 0, 496, 0, 802, 448, 634, 178, 0, 0, 0, 0, 176, 0, 712, 0, 0 for RFX3 + eGFP + cells were analyzed for AAV1, AAV2, MyoAAV 2A, AAV2-retro, AAV3b, AAV4, MyoAAV 4A, AAV5, AAV6, AAV6.2, AAV7m8, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8, AAVie, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, and 3838, 3808, 5224, 5976, 6526, 2280, 4572, 2978, 1818, 2328, 3848, 2734, 2602, 2800, 4156, 5136, 3778, 4634, 4940, 4376 for RFX3 + cells, respectively. n = 3. c Ratio of Acetyl-α-Tubulin + cells that are eGFP + .778, 0, 0, 0, 0, 642, 0, 822, 562, 666, 174, 0, 0, 0, 0, 178, 0, 616, 0, 0 for Acetyl-α-Tubulin + eGFP + cells were analyzed for AAV1, AAV2, MyoAAV 2A, AAV2-retro, AAV3b, AAV4, MyoAAV 4A, AAV5, AAV6, AAV6.2, AAV7m8, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8, AAVie, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, and 4704, 4590, 6018, 6834, 8024, 2930, 5508, 3204, 2320, 2634, 3974, 3672, 5916, 5032, 5270, 5780, 4216, 4090, 4046, 5678 for Acetyl-α-Tubulin + cells, respectively. n = 3. d Ratio of HOPX + cells that are eGFP + . 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 78, 0, 132, 0, 0, 0, 0, 0, 0 for HOPX + eGFP + cells were analyzed for AAV1, AAV2, MyoAAV 2A, AAV2-retro, AAV3b, AAV4, MyoAAV 4A, AAV5, AAV6, AAV6.2, AAV7m8, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8, AAVie, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, and 1470, 3780, 2478, 3136, 3234, 2828, 2226, 4074, 2996, 3052, 3780, 2898, 2772, 3606, 3262, 2142, 1778, 1148, 1890, 1302 for HOPX + cells, respectively. n = 3. e Ratio of SFTPC + cells that are eGFP + . 1020, 408, 0, 252, 444, 766, 0, 2242, 2534, 2330, 1668, 1512, 1054, 1040, 950, 1144, 2142, 662, 860, 490 for SFTPC + eGFP + cells were analyzed for AAV1, AAV2, MyoAAV 2A, AAV2-retro, AAV3b, AAV4, MyoAAV 4A, AAV5, AAV6, AAV6.2, AAV7m8, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8, AAVie, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, and 1586, 3782, 2478, 4250, 6468, 1568, 2226, 4070, 3582, 3348, 9194, 5282, 5366, 5340, 3070, 3260, 3728, 2436, 2600, 2120 for SFTPC + cells, respectively. n = 3. f Ratio of ERG + cells that are eGFP + . 66, 58, 0, 72, 0, 52, 0, 96, 98, 76, 0, 78, 0, 0, 76, 76, 114, 122, 64, 70 for ERG + eGFP + cells were analyzed for AAV1, AAV2, MyoAAV 2A, AAV2-retro, AAV3b, AAV4, MyoAAV 4A, AAV5, AAV6, AAV6.2, AAV7m8, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8, AAVie, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, and 8364, 19,142, 10,392, 22,746, 13,582, 12,748, 9490, 16,240, 26,052, 19,534, 11,782, 20,028, 8488, 8262, 16,880, 14,944, 15,482, 11,352, 10,870, 9828 for ERG + cells, respectively. n = 3

Article Snippet: AAVs serotypes (PackGene Biotech) used in this study were as follows: AAV1-CAG-Dio-eGFP-WPRE-pA, AAV2-CAG-Dio-eGFP-WPRE-pA, MyoAAV 2A-CAG-Dio-eGFP-WPRE-pA, AAV2-retro-CAG-Dio-eGFP-WPRE-pA, AAV3b-CAG-Dio-eGFP-WPRE-pA, AAV4-CAG-Dio-eGFP-WPRE-pA, MyoAAV 4A-CAG-Dio-eGFP-WPRE-pA, AAV5-CAG-Dio-eGFP-WPRE-pA, AAV6-CAG-Dio-eGFP-WPRE-pA, AAV6.2-CAG-Dio-eGFP-WPRE-pA, AAV7m8-CAG-Dio-eGFP-WPRE-pA, AAV8-CAG-Dio-eGFP-WPRE-pA, AAV9-CAG-Dio-eGFP-WPRE-pA, AAVrh10-CAG-Dio-eGFP-WPRE-pA, AAV-DJ-CAG-Dio-eGFP-WPRE-pA, AAV-DJ/8-CAG-Dio-eGFP-WPRE-pA, AAV-ie-CAG-Dio-eGFP-WPRE-pA, AAV-PHP.B-CAG-Dio-eGFP-WPRE-pA, AAV-PHP.eB-CAG-Dio-eGFP-WPRE-pA, and AAV-PHP.S-CAG-Dio-eGFP-WPRE-pA. For each mouse, 40 μL of AAV-containing solution (10 [ ] GC/mL) was injected into the lungs through the trachea.

Techniques: Infection

Journal: bioRxiv

Article Title: Generalized fear following acute stress is caused by change in co-transmitter identity of serotonergic neurons

doi: 10.1101/2023.05.10.540268

Figure Lengend Snippet: ( A ) Experimental design to identify presynaptic changes in transporter expression of serotonergic lwDR axons. AAV8-phSyn1-FLEX-tdTomato-T2A-Syptophysin-EGFP-WPRE (AAV-FLEX-TdTomato-T2A-SypEGFP) was injected into the lwDR of SERT-Cre mice. Coronal sections containing the CeA, LHA, LHB or PVN were immunostained for EGFP and VGLUT3 or EGFP and VGAT. ( B ) Double immunostaining of TdTomato and 5-HT in the lwDR of mice that received 1.5 mA shock. ( C, D ) Double immunostaining of VGLUT3 or VGAT and EGFP in the CeA of shocked or control mice injected with AAV-FLEX-SypEGFP-T2A-TdTomato in the lwDR. Scale bar, 10 μm. ( E, F ) The M1 coefficient of VGLUT3 or VGAT colocalization with putative serotonergic presynaptic terminals. N>800 EGFP+ terminals/mouse; n=3 mice/group. ( G ) Experimental design to validate the targets of 5-HT lwDR neurons that have switched from co-expressing VGLUT3 to coexpressing GAD67. ( H ) Quintuple-labeling of GAD67, VGLUT3, fluorogold, mCherry and 5-HT in the lwDR of both control and shocked mice. Scale bar, 10 μm. ( I ) Representative coronal sections show fluorogold (white) injected into the CeA or LHA. Scale bar, 1 mm. ( J ) Percent of neurons expressing 5-HT, mCherry and fluorogold that express GAD67 but not VGLUT3. The x-axis indicates the brain regions in which fluorogold was injected. n=3 mice/group. ( K ) Experimental design to test whether blocking the gain of GAD67 in 5-HT lwDR neurons that project to the CeA or LHA suppresses generalized fear. ( L ) Conditioned and ( M ) generalized fear of mice injected in ( K ), housed for 4 weeks, shocked at 1.5 mA and tested two weeks later. n=5-8 mice/group. ( N ) Cartoon showing the switch from VGLUT3 to GAD67 in the serotonergic cell bodies is consistent with a switch in transporter expression from VGLUT3 to VGAT at serotonergic terminals in the CeA and LHA.

Article Snippet: Vector Biolabs produced AAV8-CAG-DIO-shRNAmir-mGAD1-EGFP ( , ), AAV8-CAG-DIO-shRNAmir-mVGLUT3-EGFP, AAV8-CAG-DIO-shRNAmir-Scramble-EGFP, AAV8-CAG-shRNAmir-GR-EGFP, and AAV8-CAG-shRNAmir-Scramble-EGFP.

Techniques: Expressing, Injection, Double Immunostaining, Labeling, Blocking Assay

Journal: bioRxiv

Article Title: Generalized fear following acute stress is caused by change in co-transmitter identity of serotonergic neurons

doi: 10.1101/2023.05.10.540268

Figure Lengend Snippet: ( A ) Experimental design to identify presynaptic changes in transporter expression of serotonergic lwDR axons. AAV8-phSyn1-FLEX-tdTomato-T2A-Syptophysin-EGFP-WPRE (AAV-FLEX-TdTomato-T2A-SypEGFP) was injected into the lwDR of SERT-Cre mice. Coronal sections containing the CeA, LHA, LHB or PVN were immunostained for EGFP and VGLUT3 or EGFP and VGAT. ( B ) Double immunostaining of TdTomato and 5-HT in the lwDR of mice that received 1.5 mA shock. ( C, D ) Double immunostaining of VGLUT3 or VGAT and EGFP in the CeA of shocked or control mice injected with AAV-FLEX-SypEGFP-T2A-TdTomato in the lwDR. Scale bar, 10 μm. ( E, F ) The M1 coefficient of VGLUT3 or VGAT colocalization with putative serotonergic presynaptic terminals. N>800 EGFP+ terminals/mouse; n=3 mice/group. ( G ) Experimental design to validate the targets of 5-HT lwDR neurons that have switched from co-expressing VGLUT3 to coexpressing GAD67. ( H ) Quintuple-labeling of GAD67, VGLUT3, fluorogold, mCherry and 5-HT in the lwDR of both control and shocked mice. Scale bar, 10 μm. ( I ) Representative coronal sections show fluorogold (white) injected into the CeA or LHA. Scale bar, 1 mm. ( J ) Percent of neurons expressing 5-HT, mCherry and fluorogold that express GAD67 but not VGLUT3. The x-axis indicates the brain regions in which fluorogold was injected. n=3 mice/group. ( K ) Experimental design to test whether blocking the gain of GAD67 in 5-HT lwDR neurons that project to the CeA or LHA suppresses generalized fear. ( L ) Conditioned and ( M ) generalized fear of mice injected in ( K ), housed for 4 weeks, shocked at 1.5 mA and tested two weeks later. n=5-8 mice/group. ( N ) Cartoon showing the switch from VGLUT3 to GAD67 in the serotonergic cell bodies is consistent with a switch in transporter expression from VGLUT3 to VGAT at serotonergic terminals in the CeA and LHA.

Article Snippet: Vector Biolabs produced AAV8-CAG-DIO-shRNAmir-mGAD1-EGFP ( , ), AAV8-CAG-DIO-shRNAmir-mVGLUT3-EGFP, AAV8-CAG-DIO-shRNAmir-Scramble-EGFP, AAV8-CAG-shRNAmir-GR-EGFP, and AAV8-CAG-shRNAmir-Scramble-EGFP.

Techniques: Expressing, Injection, Double Immunostaining, Labeling, Blocking Assay

Journal: bioRxiv

Article Title: Generalized fear following acute stress is caused by change in co-transmitter identity of serotonergic neurons

doi: 10.1101/2023.05.10.540268

Figure Lengend Snippet: ( A ) Experimental design to identify presynaptic changes in transporter expression of serotonergic lwDR axons. AAV8-phSyn1-FLEX-tdTomato-T2A-Syptophysin-EGFP-WPRE (AAV-FLEX-TdTomato-T2A-SypEGFP) was injected into the lwDR of SERT-Cre mice. Coronal sections containing the CeA, LHA, LHB or PVN were immunostained for EGFP and VGLUT3 or EGFP and VGAT. ( B ) Double immunostaining of TdTomato and 5-HT in the lwDR of mice that received 1.5 mA shock. ( C, D ) Double immunostaining of VGLUT3 or VGAT and EGFP in the CeA of shocked or control mice injected with AAV-FLEX-SypEGFP-T2A-TdTomato in the lwDR. Scale bar, 10 μm. ( E, F ) The M1 coefficient of VGLUT3 or VGAT colocalization with putative serotonergic presynaptic terminals. N>800 EGFP+ terminals/mouse; n=3 mice/group. ( G ) Experimental design to validate the targets of 5-HT lwDR neurons that have switched from co-expressing VGLUT3 to coexpressing GAD67. ( H ) Quintuple-labeling of GAD67, VGLUT3, fluorogold, mCherry and 5-HT in the lwDR of both control and shocked mice. Scale bar, 10 μm. ( I ) Representative coronal sections show fluorogold (white) injected into the CeA or LHA. Scale bar, 1 mm. ( J ) Percent of neurons expressing 5-HT, mCherry and fluorogold that express GAD67 but not VGLUT3. The x-axis indicates the brain regions in which fluorogold was injected. n=3 mice/group. ( K ) Experimental design to test whether blocking the gain of GAD67 in 5-HT lwDR neurons that project to the CeA or LHA suppresses generalized fear. ( L ) Conditioned and ( M ) generalized fear of mice injected in ( K ), housed for 4 weeks, shocked at 1.5 mA and tested two weeks later. n=5-8 mice/group. ( N ) Cartoon showing the switch from VGLUT3 to GAD67 in the serotonergic cell bodies is consistent with a switch in transporter expression from VGLUT3 to VGAT at serotonergic terminals in the CeA and LHA.

Article Snippet: Vector Biolabs produced AAV8-CAG-DIO-shRNAmir-mGAD1-EGFP ( , ), AAV8-CAG-DIO-shRNAmir-mVGLUT3-EGFP, AAV8-CAG-DIO-shRNAmir-Scramble-EGFP, AAV8-CAG-shRNAmir-GR-EGFP, and AAV8-CAG-shRNAmir-Scramble-EGFP.

Techniques: Expressing, Injection, Double Immunostaining, Labeling, Blocking Assay