Journal: Pathogens

Article Title: Evaluation of RNA Secondary Stem-Loop Structures in the UTRs of Mouse Hepatitis Virus as New Therapeutic Targets

doi: 10.3390/pathogens13060518

Figure Lengend Snippet: Design of RNAi targets in 5′ and 3′ UTRs in MHV-A59 genome. ( a ) The sequence schematic diagram to display RNAi (shRNA and siRNA) targets in RNA secondary SL structures of MHV-A59 5′ UTR. ( b ) The sequence schematic diagram to display RNAi (shRNA and siRNA) targets in RNA secondary SL structures of MHV-A59 3′ UTR. In a previous study , the removal of SL1 A35 (5′ΔA35) in the MHV-A59 5′ untranslated region (UTR) resulted in changes to A31307G (3′A29G, numbered from the 3′ end) and A31257G (3′A78G, numbered from the 3′ end) in the MHV-A59 3′ UTR. The RNA secondary SL structures were labelled 5′ΔA35, 3’A29G, and 3’A78G, respectively.

Article Snippet: Furthermore, the full sequences of MHV-A59 5′UTR and 3′UTR were applied to VectorBuilder’s shRNA target design tool ( https://www.vectorbuilder.kr/tool/shrna-target-design.html , accessed on 1 January 2022).

Techniques: Sequencing, shRNA

Journal: Pathogens

Article Title: Evaluation of RNA Secondary Stem-Loop Structures in the UTRs of Mouse Hepatitis Virus as New Therapeutic Targets

doi: 10.3390/pathogens13060518

Figure Lengend Snippet: Inhibitory effects of shRNAs targeting RNA secondary SL structures in the 5′UTR on MHV-A59 replication. ( a ) RNA dot blot analysis for inhibitory effects of shRNA on MHV replication. DBT cells (DBT-5′shRNA cells) expressing shRNAs (5′shRNA) targeted for MHV-A59 5′UTR SL structures were established by the shRNA lentiviral system. MHV-A59 was infected at an M.O.I 10 −1 to 10 −4 into DBT-5′shRNA cells. RNA dot blot probes were made to detect the MHV N gene (NC_048217.1, nt 31,519-31,018). ( b ) As an internal control, the expression of the actin gene (NM_007393.5, nt 668-1180) in DBT-5′shRNA cells was analyzed by RNA dot blot assay. ( c ) The relative inhibitory effect of 5′shRNAs on MHV-A59 infection based on actin expression was quantified by ImageJ software ( https://imagej.net/ij/ , accessed on 1 January 2023) . One-way analysis of variance was performed, followed by Tukey’s multiple comparison test to verify statistical significance by 5′shRNAs. Statistically significant groups were labeled as A, B, and C to differentiate them. ( d ) Northern analysis for inhibitory effect of 5′shRNA on the MHV sgRNA synthesis. MHV-A59 at an M.O.I of 0.5 was infected into DBT-5′shRNA cells for 24 h. Mock (negative control) was performed with DBT-5′shRNA cells that were not infected with MHV-A59. Patterns of the MHV-A59 sgRNA production in DBT-5′shRNA cells were investigated with a DIG labelled N gene probe. ( e ) The effect of 5′shRNA inhibition on the MHV sgRNA synthesis was quantitatively analyzed with Image J software. ( f ) The one-step growth experiments of MHV-A59 infected DBT-5′shRNA cells. 5 × 10 4 DBT cells were seeded in 24-well plates and infected with MHV-A59 at an M.O.I of 3 for 0, 6, 12, 18, and 24 h the next day. After each infection time, MHV-A59 was harvested and the growth rate of the virus was analyzed. ( g ) Representative plaque shape formed by MHV-A59 infecting DBT-5′shRNA cells. ( h ) Average plaque size of MHV-A59 formed on DBT-5′shRNA cells. Statistical analysis was performed using a student’s t -test and a one-way analysis of variance (ANOVA). A post-hoc test was conducted using Tukey’s multiple comparisons test. ( i ) Analysis of the 5′UTR sequence of MHV-A59 infected DBT-5′shRNA cells. The MHV-A59 virus, harvested after infecting DBT-5′shRNA cells, was designated as P0. Subsequently, the harvested virus obtained after re-infecting cells was named P1, and the virus obtained after another round of infection and harvest was named P2. RNA was extracted from each of these virus samples, and sequence analysis was performed. * indicates a statistical difference in the t -test between the shCon-treated plaque size group and the shSL1/2 plaque size group; ** indicates a statistical difference in the t -test between the shCon-treated plaque size group and the shSL3/4 plaque size group; *** indicates a statistical difference in the t -test statistical difference between the shCon-treated plque size group and the siMIN-treated plque size group is significant.

Article Snippet: Furthermore, the full sequences of MHV-A59 5′UTR and 3′UTR were applied to VectorBuilder’s shRNA target design tool ( https://www.vectorbuilder.kr/tool/shrna-target-design.html , accessed on 1 January 2022).

Techniques: Dot Blot, shRNA, Expressing, Infection, Control, Software, Comparison, Labeling, Northern Blot, Negative Control, Inhibition, Virus, Sequencing

Journal: Pathogens

Article Title: Evaluation of RNA Secondary Stem-Loop Structures in the UTRs of Mouse Hepatitis Virus as New Therapeutic Targets

doi: 10.3390/pathogens13060518

Figure Lengend Snippet: Inhibitory effects of siRNAs targeting RNA secondary SL structures in the UTR on MHV replication. ( a ) RNA dot blot analysis for inhibitory effects of 5′siRNA on MHV replication. DBT cells (DBT-5′siRNA cells) were treated with two concentrations (100, 500 ng) of siRNA (5′siRNA) targeted for MHV-A59 5′UTR SL structures. DBT-5′siRNA cells were infected with MHV-A59 at an M.O.I of 10 −2 for 24 h. RNA dot blot probes were made to detect the MHV N gene. ( b ) As an internal control, the expression of the actin gene in DBT-5′siRNA cells was analyzed by RNA dot blot assay. ( c ) The relative inhibitory effect of siRNAs on MHV-A59 infection based on actin expression was quantified by Image J software. One-way analysis of variance was performed, followed by Tukey’s multiple comparison test to verify statistical significance by 5′siRNAs. Statistically significant groups were labeled as A, B, C, D, E, F and G to differentiate them. ( d ) Northern analysis for inhibitory effect of 20 nM siSL1 on the MHV sgRNA synthesis. MHV-A59 at an M.O.I of 0.5 was infected into DBT-5′siRNA cells for 24 h. Mock (negative control) was performed with DBT-5′siRNA cells that were not infected with MHV-A59. Patterns of MHV-A59 sgRNA production were investigated with a DIG labelled N gene probe. ( e ) The effect of 20 nM siSL1 inhibition on the MHV sgRNA synthesis was quantitatively analyzed with Image J software. ( f ) The one-step growth curve of MHV-A59-infected DBT-5′siRNA cells treated with 20 nM siSL1. 5 × 10 4 DBT cells were seeded in 24-well plates and infected with MHV-A59 at an M.O.I of 3 for 0, 6, 12, 18, and 24 h the next day. After each infection time, MHV-A59 was harvested and the growth rate of the virus was analyzed. ( g ) Representative plaque shape formed by MHV-A59 infecting DBT-siRNA cells treated with 20 nM siSL1. ( h ) Average plaque size of MHV-A59 formed on DBT-5′siRNA cells treated with 20 nM siSL1. Statistical analysis was performed using a student’s t -test and a one-way analysis of variance (ANOVA). A post-hoc test was conducted using Tukey’s multiple comparisons test. ( i ) Analysis of the 5′UTR sequence of MHV-A59-infected DBT-5′siRNA cells treated with 20 nM siSL1. The MHV-A59 virus, harvested after infecting DBT-5′siRNA cells, was designated as P0. Subsequently, the harvested virus obtained after re-infecting cells was named P1, and the virus obtained after another round of infection and harvest was named P2. RNA was extracted from each of these virus samples, and sequence analysis was performed. ( j ) Detection of MHV-A59 gRNA in DBT cells treated with 10 nM siSL1. MHV-A59 negative-strand RNA and positive-strand gRNA were quantified relative to actin control by RT-qPCR assay. The negative-strand RNA synthesis in siSL1-treated DBT cells was statistically significantly decreased compared to the negative-strand RNA synthesis in siCon-treated DBT cells. Statistically significant groups were labeled as A and B to differentiate them. * indicates a statistical difference in the t -test between the shCon-treated plaque size group and the shSL1/2 plaque size group; ** indicates a statistical difference in the t -test between the shCon-treated plaque size group and the shSL3/4 plaque size group.

Article Snippet: Furthermore, the full sequences of MHV-A59 5′UTR and 3′UTR were applied to VectorBuilder’s shRNA target design tool ( https://www.vectorbuilder.kr/tool/shrna-target-design.html , accessed on 1 January 2022).

Techniques: Dot Blot, Infection, Control, Expressing, Software, Comparison, Labeling, Northern Blot, Negative Control, Inhibition, Virus, Sequencing, Quantitative RT-PCR

Journal: Pathogens

Article Title: Evaluation of RNA Secondary Stem-Loop Structures in the UTRs of Mouse Hepatitis Virus as New Therapeutic Targets

doi: 10.3390/pathogens13060518

Figure Lengend Snippet: Inhibitory effects of shRNAs targeting RNA secondary SL structures in the 3′UTR on MHV replication. ( a ) RNA dot blot analysis for inhibition of MHV replication by 3′sh1 and 3′sh2 targeting MHV-A59 3′UTR pseudoknot. DBT-3′sh1 and DBT-3′sh2 cells expressing 3′sh1 and 3′sh2 were established by the shRNA lentiviral system. MHV-A59 was infected at an M.O.I of 10 −1 to 10 −4 into DBT-3′sh1 and DBT-3′sh2 cells, respectively. RNA dot blot probes were made to detect the MHV N gene. ( b ) As an internal control, the expression of the actin gene in DBT-3′sh1 and DBT-3′sh2 cells was analyzed by RNA dot blot assay, respectively. ( c ) The relative inhibitory effect of 3′sh1 and 3′sh2 on MHV-A59 infection based on actin expression was quantified by Image J software, respectively. MHV-A59 N gene expression was evaluated relative to actin gene expression in DBT-3′sh1 and DBT-3′sh2 cells infected with MHV-A59, respectively. One-way analysis of variance was performed, followed by Tukey’s multiple comparison test to verify statistical significance by 3′shRNAs. Statistically significant groups were labeled as A and B to differentiate them. ( d ) RNA dot blot analysis for inhibition of MHV replication by 3′sh3 and 3′sh4 targeting MHV-A59 3′UTR pseudoknot. DBT-3′sh3 and DBT-3′sh4 cells expressing 3′sh3 and 3′sh4 were established by the shRNA lentiviral system. MHV-A59 was infected at an M.O.I of 10 −1 to 10 −4 into DBT-3′sh3 and DBT-3′sh4 cells, respectively. RNA dot blot probes were made to detect the MHV N gene. ( e ) As an internal control, the expression of the actin gene in DBT-3′sh3 and DBT-3′sh4 cells was analyzed by RNA dot blot assay, respectively. ( f ) The relative inhibitory effect of 3′sh3 and 3′sh4 on MHV-A59 infection based on actin expression was quantified by Image J software, respectively. MHV-A59 N gene expression was evaluated relative to actin gene expression in DBT-3′sh3 and DBT-3′sh4 cells infected with MHV-A59, respectively. One-way analysis of variance was performed, followed by Tukey’s multiple comparison test to verify statistical significance by 3′shRNAs. Statistically significant groups were labeled as A, B and C to differentiate them. ( g ) Northern analysis for inhibitory effect of 3′sh1 on the MHV sgRNA synthesis. MHV-A59 at an M.O.I of 0.5 was infected into DBT-3′sh1 cells for 24 h. Mock (negative control) was performed with DBT-3′sh1 cells that were not infected with MHV-A59. MHV sgRNA production pattern was probed with a DIG labelled N gene probe. ( h ) The effect of 3′sh1 inhibition on MHV sgRNA production was quantitatively analyzed with Image J software. ( i ) Northern analysis for inhibitory effect of 3′sh2 on MHV sgRNA synthesis. MHV-A59 at an M.O.I of 0.5 was infected into DBT-3′sh2 cells for 24 h. Mock (negative control) was performed with DBT-3′sh2 cells that were not infected with MHV-A59. MHV sgRNA synthesis pattern was probed with a DIG labelled N gene probe. ( j ) The effect of 3′sh2 inhibition on the MHV sgRNA synthesis was quantitatively analyzed with Image J software. ( k ) The one-step growth experiments of MHV-A59-infected DBT-3′sh1 cells. 5 × 10 4 DBT cells were seeded in 24-well plates and infected with MHV-A59 at an M.O.I of 3 for 0, 6, 12, 18, and 24 h the next day. After each infection time, MHV-A59 was harvested and the growth rate of the virus was analyzed. ( l ) Representative plaque shape formed by MHV-A59 infecting DBT-3′sh1 cells. ( m ) Average plaque size of MHV-A59 formed on DBT-3′sh1 cells. Statistical analysis was performed using a student’s t -test and a one-way analysis of variance (ANOVA). A post-hoc test was conducted using Tukey’s multiple comparisons test. * indicates a statistical difference in the t -test between the shCon-treated plaque size group and the shSL1/2 plaque size group; ** indicates a statistical difference in the t -test between the shCon-treated plaque size group and the shSL3/4 plaque size group; *** indicates a statistical difference in the t -test statistical difference between the shCon-treated plque size group and the siMIN-treated plque size group is significant.

Article Snippet: Furthermore, the full sequences of MHV-A59 5′UTR and 3′UTR were applied to VectorBuilder’s shRNA target design tool ( https://www.vectorbuilder.kr/tool/shrna-target-design.html , accessed on 1 January 2022).

Techniques: Dot Blot, Inhibition, Expressing, shRNA, Infection, Control, Software, Gene Expression, Comparison, Labeling, Northern Blot, Negative Control, Virus

Journal: Pathogens

Article Title: Evaluation of RNA Secondary Stem-Loop Structures in the UTRs of Mouse Hepatitis Virus as New Therapeutic Targets

doi: 10.3390/pathogens13060518

Figure Lengend Snippet: Inhibitory effect of siRNAs targeting RNA secondary SL structures in the 3′UTR on MHV replication. ( a ) RNA dot blot analysis for inhibitory effect of 3′siRNA on MHV replication. DBT cells (DBT-3′siRNA cells) were treated with two concentrations (100, 500 ng) of siRNA (3′siRNA) targeted for MHV-A59 3′UTR SL structures. DBT-3′siRNA cells were infected with MHV-A59 at an M.O.I of 10 −2 for 24 h. RNA dot blot probes were made to detect the MHV N gene. ( b ) As an internal control, the expression of the actin gene in DBT-3′siRNA cells was analyzed by RNA dot blot assay. ( c ) The relative inhibitory effect of 3′siRNAs on MHV-A59 infection based on actin expression was quantified by Image J software. MHV-A59 N gene expression was evaluated relative to actin gene expression in DBT-3′siRNA cells infected with MHV-A59. One-way analysis of variance was performed, followed by Tukey’s multiple comparison test to verify statistical significance by 3′siRNAs. Statistically significant groups were labeled as A, B, C, D, E and F to differentiate them. ( d ) Northern analysis for inhibitory effect of 20 nM siMIN on the MHV sgRNA synthesis. MHV-A59 at an M.O.I of 0.5 was infected into DBT-3′siRNA cells for 24 h. Mock (negative control) was performed with DBT-3′siRNA cells that were not infected with MHV-A59. Patterns of MHV-A59 sgRNA production in DBT-3′siRNA cells were investigated with a DIG labelled N gene probe. ( e ) The effect of siMIN inhibition on the MHV sgRNA synthesis was quantitatively analyzed with Image J software. (f) The one-step growth experiments of MHV-A59-infected DBT-3′siRNA cells treated with 20 nM siMIN. 5 × 10 4 DBT cells were seeded in 24-well plates and infected with MHV-A59 at an M.O.I of 3 for 0, 6, 12, 18, and 24 h the next day. After each infection time, MHV-A59 was harvested and the growth rate of the virus was analyzed. ( g ) Representative plaque shape formed by MHV-A59 infecting DBT-3′siRNA cells treated with 20 nM siMIN. ( h ) Average plaque size of MHV-A59 formed on DBT-3′siRNA cells treated with 20 nM siMIN. Statistical analysis was performed using a student’s t -test and a one-way analysis of variance (ANOVA). A post-hoc test was conducted using Tukey’s multiple comparisons test. ( i ) Analysis of 3′UTR sequence of MHV-A59 infected DBT-3′siRNA cells treated with 20 nM siMIN. The MHV-A59 virus, harvested after infecting DBT-3′siRNA cells, was designated as P0. Subsequently, the harvested virus obtained after re-infecting cells was named P1, and the virus obtained after another round of infection and harvest was named P2. RNA was extracted from each of these virus samples, and sequence analysis was performed. ( j ) Detection of MHV-A59 gRNA in DBT cells treated with 10 nM siMIN. MHV-A59 negative-strand RNA and positive-strand gRNA were quantified relative to actin control by RT-qPCR assay. The negative-strand RNA synthesis in siMIN-treated DBT cells was statistically significantly decreased compared to the negative-strand RNA synthesis in siCon-treated DBT cells. Statistically significant groups were labeled as A and B to differentiate them. * indicates a statistical difference in the t -test between the shCon-treated plaque size group and the shSL1/2 plaque size group; ** indicates a statistical difference in the t -test between the shCon-treated plaque size group and the shSL3/4 plaque size group; *** indicates a statistical difference in the t -test statistical difference between the shCon-treated plque size group and the siMIN-treated plque size group is significant.

Article Snippet: Furthermore, the full sequences of MHV-A59 5′UTR and 3′UTR were applied to VectorBuilder’s shRNA target design tool ( https://www.vectorbuilder.kr/tool/shrna-target-design.html , accessed on 1 January 2022).

Techniques: Dot Blot, Infection, Control, Expressing, Software, Gene Expression, Comparison, Labeling, Northern Blot, Negative Control, Inhibition, Virus, Sequencing, Quantitative RT-PCR

Journal: Pathogens

Article Title: Evaluation of RNA Secondary Stem-Loop Structures in the UTRs of Mouse Hepatitis Virus as New Therapeutic Targets

doi: 10.3390/pathogens13060518

Figure Lengend Snippet: Inhibitory effects of siRNAs targeting SL structures in the 5′UTR and 3′UTR of MHV-A59. ( a ) RNA dot blot assay to analyze the effectiveness of the combination of siSL1 (5′siRNA) and siMIN (siRNA) to inhibit MHV-A59 replication. DBT cells (DBT-5′3′siRNA cells) were treated with two concentrations of siSL1 and siMIN combinations. For siSL1 and siMIN monotherapy, concentrations of 10 nM and 20 nM of each siRNA were used, respectively. For combination therapy, a half-and-half mixture of the two siRNAs at concentrations of 10 nM (5 nM siSL1 + 5 nM siMIN) and 20 nM (10 nM siSL1 + 10 nM siMIN) was used. DBT-5′3′siRNA cells were infected with MHV-A59 at an M.O.I of 10 −2 for 24 h. RNA dot blot probes were made to detect the MHV N gene. ( b ) As an internal control, the expression of the actin gene in DBT-5′3′siRNA cells was analyzed by RNA dot blot assay. ( c ) The inhibitory effect of MHV-A59 replication by the combination therapy of siSL1 and siMIN (5 + 5, 10 + 10) was quantitatively analyzed by Image J software. MHV-A59 N gene expression was evaluated relative to actin gene expression in DBT-5′3′siRNA cells infected with MHV-A59. A primary one-way analysis of variance, followed by a secondary Tukey’s multiple comparison test was performed to determine the statistical significance between siSL1, siMIN, and the combination. Statistically significant groups were labeled as A, B and C to differentiate them. ( d ) Northern analysis for inhibitory effect of the combination therapy of siSL1 and siMIN (5 + 5) on the MHV sgRNA synthesis. MHV-A59 at an M.O.I of 0.5 was infected into DBT-5′3′siRNA cells for 24 h. Mock (negative control) was performed with DBT-5′3′siRNA cells that were not infected with MHV-A59. Patterns of MHV-A59 sgRNA production in DBT-5′3′siRNA cells were investigated with a DIG labelled N gene probe. ( e ) The inhibitory effect of the combination therapy of siSL1 and siMIN (5 + 5) on the MHV sgRNA synthesis was quantitatively analyzed with Image J software. Based on the sgRNA production of MHV-A59 infecting siCon-treated DBT cells, we quantified the sgRNA production of MHV-A59 infecting siSL1, si3MIN, and the combination-treated DBT cells. ( f ) The one-step growth experiments of MHV-A59-infected DBT-5′3′siRNA cells treated with the combination therapy of 5 nM siSL1 and 5 nM siMIN (5 + 5). 5 × 10 4 DBT cells were seeded in 24-well plates and infected with MHV-A59 at an M.O.I of 3 for 0, 6, 12, 18, and 24 h the next day. After each infection time, MHV-A59 was harvested and the growth rate of the virus was analyzed. ( g ) Representative plaque shapes formed by MHV-A59 infecting DBT-5′3′siRNA cells treated with the combination therapy of 5 nM siSL1 and 5 nM siMIN (5 + 5). ( h ) Average plaque size of MHV-A59 formed on DBT-5′3′siRNA cells treated with the combination therapy of 5 nM siSL1 and 5 nM siMIN (5 + 5). Statistical analysis was performed using a student’s t -test and a one-way analysis of variance (ANOVA). A post-hoc test was conducted using Tukey’s multiple comparisons test. Statistically significant groups were labeled as A and B to differentiate them.

Article Snippet: Furthermore, the full sequences of MHV-A59 5′UTR and 3′UTR were applied to VectorBuilder’s shRNA target design tool ( https://www.vectorbuilder.kr/tool/shrna-target-design.html , accessed on 1 January 2022).

Techniques: Dot Blot, Infection, Control, Expressing, Software, Gene Expression, Comparison, Labeling, Northern Blot, Negative Control, Virus

Journal: The European Journal of Neuroscience

Article Title: Integrin α5β1 is necessary for regulation of radial migration of cortical neurons during mouse brain development

doi: 10.1111/j.1460-9568.2009.07072.x

Figure Lengend Snippet: Analysis of the expression and knockdown efficiency of α5β1 integrin in the developing murine neocortex. (A) In situ hybridization of coronal brain sections at E14.5 revealed a signal in the VZ/SVZ for the α5 subunit; the signal for the β1 subunit was widespread throughout the laminar structure of the cerebral cortex and visible in the VZ and SVZ in some areas. Scale bar, 500 μm. (B, C) pcDNA3.1 vector containing an α5 (B) or α6 (C) integrin murine cDNA was cotransfected into HeLa cells with pRNAT-U6.1/Neo empty vector, the control construct (Ctr), or two different shRNA constructs targeting mouse α5 integrin (R1 and R7). Forty-eight hours after transfection, cells were lysed and were subjected to immunoblotting for α5 integrin or α6 integrin. GAPDH and GFP are, respectively, loading and transfection efficiency controls. α5-shRNA constructs were able to decrease α5 integrin expression by ∼80% (R1) and ∼40% (R7) but had no detectable effect on expression of the α6 integrin subunit. (D) High magnification of the VZ of E18.5 brain sections after electroporation with R1 α5-shRNA. Immunostaining for α5 integrin (red) and GFP (green) showed a reduction of endogenous α5 integrin expression in the cortical neuronal progenitors compared with the control brain. Scale bar, 50 μm. VZ/SVZ, ventricular zone/subventricular zone; IZ, intermediate zone; CP, cortical plate.

Article Snippet: α5 integrin target sequences were designed via the GenScript shRNA design tool and are as follows: shRNA-1 (R1), 5′-CCTGCTACCTCTCCACAGAAA-3′; shRNA-4 (R4), 5′-GCAGATCTCGGAGTCCTATTA-3′; shRNA-7 (R7), 5′-CTGCCTCAATGCCTCTGGAAA-3′; shRNA-9 (R9), 5′-ACTTTCAGATCCTCAGCAAGA-3′; control scrambled shRNA (Ctr), 5′-CACAATATCTGCCCCGATCCA-3′. shRNA constructs were generated in the pRNAT-U6.1/Neo vector (GenScript, Paris, France), which allows expression of a coral green fluorescent protein (GFP) marker with the shRNA of interest.

Techniques: Expressing, In Situ Hybridization, Plasmid Preparation, Construct, shRNA, Transfection, Western Blot, Electroporation, Immunostaining

Journal: The European Journal of Neuroscience

Article Title: Integrin α5β1 is necessary for regulation of radial migration of cortical neurons during mouse brain development

doi: 10.1111/j.1460-9568.2009.07072.x

Figure Lengend Snippet: Effect of α5 integrin knockdown in developing murine cerebral cortex. (A) Representative coronal sections of embryonic murine neocortex 3 days (E18.5) following electroporation of empty vector, Ctr, R1 and R7 α5 shRNAs together with coral GFP. Transfection of R1 and R7 shRNAs impaired radial migration. Scale bar, 100 μm. (B) Quantitative analyses of the distribution of GFP-positive cells in the various cortical layers. The cortical wall was subdivided, and numbers of GFP-positive cells were counted in each layer and expressed as a percentage of the total. Statistical differences were seen for each layer, comparing the distribution of GFP-positive cells between Ctr and R1 α5-shRNA. Asterisks indicate significant differences between the groups (*** P < 0.001, using Student’s t -test).

Article Snippet: α5 integrin target sequences were designed via the GenScript shRNA design tool and are as follows: shRNA-1 (R1), 5′-CCTGCTACCTCTCCACAGAAA-3′; shRNA-4 (R4), 5′-GCAGATCTCGGAGTCCTATTA-3′; shRNA-7 (R7), 5′-CTGCCTCAATGCCTCTGGAAA-3′; shRNA-9 (R9), 5′-ACTTTCAGATCCTCAGCAAGA-3′; control scrambled shRNA (Ctr), 5′-CACAATATCTGCCCCGATCCA-3′. shRNA constructs were generated in the pRNAT-U6.1/Neo vector (GenScript, Paris, France), which allows expression of a coral green fluorescent protein (GFP) marker with the shRNA of interest.

Techniques: Electroporation, Plasmid Preparation, Transfection, Migration, shRNA

Journal: The European Journal of Neuroscience

Article Title: Integrin α5β1 is necessary for regulation of radial migration of cortical neurons during mouse brain development

doi: 10.1111/j.1460-9568.2009.07072.x

Figure Lengend Snippet: Influence of α5-shRNA on radial glial scaffold and neuronal morphology. (A) Coronal sections of unelectroporated hemisphere and of brains electroporated at E15.5 with Ctr and R1 α5-shRNAs were stained with anti-Nestin (red) and GFP (green) antibodies. GFP-positive radial fibers transfected with R1 α5-shRNA appeared normal as compared with unelectroporated and Ctr α5-shRNA-transfected fibers. Scale bar, 100 μm. Bottom panels are magnified views of the MZ. The white arrows indicate basal endfeet of radial fibers attached to the pia. Transfection of R1 α5-shRNA does not seem to disturb formation of glial endfeet. MZ, marginal zone. Scale bar, 50 μm. (B) Morphology of VZ/SVZ neurons transfected with Ctr or R1 α5 shRNAs. The electroporated cells with Ctr shRNA showed a normal bipolar morphology in the premigratory region (white asterisk, high magnification). In contrast, cells expressing shRNAs against α5 integrin are arrested within the VZ/SVZ with a multipolar shape (white asterisk, high magnification). Scale bars: top, 50 μm; bottom, 10 μm.

Article Snippet: α5 integrin target sequences were designed via the GenScript shRNA design tool and are as follows: shRNA-1 (R1), 5′-CCTGCTACCTCTCCACAGAAA-3′; shRNA-4 (R4), 5′-GCAGATCTCGGAGTCCTATTA-3′; shRNA-7 (R7), 5′-CTGCCTCAATGCCTCTGGAAA-3′; shRNA-9 (R9), 5′-ACTTTCAGATCCTCAGCAAGA-3′; control scrambled shRNA (Ctr), 5′-CACAATATCTGCCCCGATCCA-3′. shRNA constructs were generated in the pRNAT-U6.1/Neo vector (GenScript, Paris, France), which allows expression of a coral green fluorescent protein (GFP) marker with the shRNA of interest.

Techniques: shRNA, Staining, Transfection, Expressing

Journal: The European Journal of Neuroscience

Article Title: Integrin α5β1 is necessary for regulation of radial migration of cortical neurons during mouse brain development

doi: 10.1111/j.1460-9568.2009.07072.x

Figure Lengend Snippet: Effect of α5-shRNA on cerebral cortex laminar structure. (A) Representative coronal sections of embryonic murine neocortex 6 days (P2) following electroporation of Ctr and R1 constructs at E15.5. Transfection of R1 induces cortical lamination defects. Scale bar, 100 μm. (B) Quantitative analyses of the distribution of GFP-positive cells in the various cortical layers. Ctip-2 (red) labels layer V. Statistical differences were seen for each layer, comparing the distribution of GFP-positive cells between Ctr and R1 constructs. Asterisks indicate significant differences between the groups (*** P < 0.001; ** P < 0.005; * P < 0.02). (C) Morphology of neurons in IZ transfected with Ctr and R1 α5 shRNAs. Cells expressing shRNAs against α5 integrin showed atypical morphology compared with control transfected cells (white arrows). Scale bar, 50 μm.

Article Snippet: α5 integrin target sequences were designed via the GenScript shRNA design tool and are as follows: shRNA-1 (R1), 5′-CCTGCTACCTCTCCACAGAAA-3′; shRNA-4 (R4), 5′-GCAGATCTCGGAGTCCTATTA-3′; shRNA-7 (R7), 5′-CTGCCTCAATGCCTCTGGAAA-3′; shRNA-9 (R9), 5′-ACTTTCAGATCCTCAGCAAGA-3′; control scrambled shRNA (Ctr), 5′-CACAATATCTGCCCCGATCCA-3′. shRNA constructs were generated in the pRNAT-U6.1/Neo vector (GenScript, Paris, France), which allows expression of a coral green fluorescent protein (GFP) marker with the shRNA of interest.

Techniques: shRNA, Electroporation, Construct, Transfection, Expressing

Journal: The Journal of Experimental Medicine

Article Title: A role for GPx3 in activity of normal and leukemia stem cells

doi: 10.1084/jem.20102386

Figure Lengend Snippet: Gpx3 depletion decreased LSC competitiveness. Left: effect of the RNAi-mediated depletion of Gpx3 on the competitiveness of FLA2 leukemic cells. 2 × 10 5 fresh FLA2 BM cells were co-cultured with shLuc or shGpx3 retrovirus-producing GP+E-86 cells. After infection (d0), cells were partitioned between flow cytometric analysis of GFP + frequency and transplantation of unsorted cells into sublethally irradiated recipients (shLuc, n = 12; shGpx3, n = 16). Contribution of GFP + (shRNA-transduced) cells to leukemia was determined on day 19. Results are shown as normalized values: the GFP + frequency in inoculum was considered 100% (dotted line, d0), and the day 19 GFP + frequency was calculated as a percentage of that value. Horizontal bars represent mean values determined for each experimental group. Middle: day 19 MFIs of GFP + cells in recipients of shLuc- and shGpx3-infected FLA2 leukemic cells. The dotted line represents MFI of GFP + cells in shLuc and shGpx3 inocula. Horizontal bars represent mean values determined for each experimental group. Right: GFP fluorescence intensity in representative recipients of shLuc- or shGpx3-infected FLA2 cells. The inset demonstrates comparable MFI GFP fluorescence in shLuc and shGpx3 inocula. *, P = 0.0037 and P = 0.0015, respectively (Wilcoxon’s test).

Article Snippet: Two shRNAs were selected as single-stranded oligonucleotides also incorporating mir-30 flanking arms using the RNAi Central shRNA psm2 design tool (G. Hannon, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Techniques: Cell Culture, Infection, Transplantation Assay, Irradiation, shRNA, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: A role for GPx3 in activity of normal and leukemia stem cells

doi: 10.1084/jem.20102386

Figure Lengend Snippet: Gpx3 regulates the self-renewal capacity of normal HSCs. (A) Gpx3 expression in HSC isolated from 3- or 4-wk-old mice (KLS SLAM cells) compared with HSC from E14.5 FL (SLAM Sca + Mac1 + ) cells. Mean ± SD, n = 2. (B) Empty vector ( n = 3), Prdm16 ( n = 4), Hoxb4 ( n = 3), NA10HD ( n = 4), and Klf10 ( n = 3) were overexpressed in Lin − CD150 + CD48 − sorted BM cells as previously described . Gpx3 mRNA was evaluated by qRT-PCR. (C) 5-FU–treated BM cells were transduced with shLuc or shGpx3. Gpx3 mRNA was evaluated by qRT-PCR in four experiments (left). CFC output was assessed in two experiments (right; mean ± SD; n = 8). GFP + cells were shRNA-transduced cells. (D) Evaluation of short-term and LTR ability of KLS CD150 + CD48 − Ly5.1 + BM cells expressing shLuc or shGpx3 in competitive transplantations into congenic Ly5.2 mice. Results are presented from both peripheral blood (PB) and BM, and represent mean ± SEM at the indicated time points for 8 mice per group. The level of gene transfer was near 100% (right) and Gpx3 silencing was obtained in all experiments ( n = 4). Two independent shGpx3 constructs (#5 and #6) were used. (E) Gpx3 , Hoxb4 , or empty vector was overexpressed in BM KLS CD150 + CD48 − sorted cells (as described in ), cells were expanded for 7 d ex vivo, and competitive transplantations in Ly5.2 congenic mice were performed. Results are shown as mean ± SEM of peripheral blood reconstitution at the indicated time points for three mice per group in five experiments. *, P = 0.0015 (Wilcoxon’s test). (F) Southern blot analyses of Gpx3 proviral integrations in DNA isolated from BM from mice reconstituted 20 wk earlier with Gpx3 -transduced cells. n = 2 experiments, three mice per experiment. (G) Analysis of differentiation potential of vector or Gpx3 -transduced cells in vivo . Blood cells were collected from recipients transplanted 20 wk earlier and stained with antibodies specific for B, T, and myeloid cells (B220, CD3, and CD11b, respectively). Values are mean ± SD (vector: n = 5; Gpx3 , n = 2).

Article Snippet: Two shRNAs were selected as single-stranded oligonucleotides also incorporating mir-30 flanking arms using the RNAi Central shRNA psm2 design tool (G. Hannon, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Techniques: Expressing, Isolation, Plasmid Preparation, Quantitative RT-PCR, Transduction, shRNA, Construct, Ex Vivo, Southern Blot, In Vivo, Staining