retroviral vector Search Results


electrodes  (KCAS Bioanalytical and Biomarker Services)
94
KCAS Bioanalytical and Biomarker Services electrodes
Electrodes, supplied by KCAS Bioanalytical and Biomarker Services, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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retroviral vector  (TaKaRa)
96
TaKaRa retroviral vector
<t>Retroviral</t> expression of Cx32 and gap junctional communication in androgen-responsive LNCaP cells. LNCaP cells were infected with the control and Cx32-harboring recombinant retroviruses, and Cx32 expression level and junctional communication were determined by Western blot (A), immunocytochemical (B, left panels) and functional (B, right panels) analyses. (A) Note that only clones infected with Cx32-harboring retroviruses (lanes LN-32-1 and LN-32-2) express Cx32 (a 27-kDa monomer and a 54-kDa dimer), whereas a control clone infected with the control retrovirus (lane LN-NEO-1) and parental, early passage cells (lane LN-WT) do not. (B) Note that Cx32-expressing LNCaP cells form gap junctions (green, bottom left panel, yellow arrows), whereas no gap junctions are detected in control cells (top left panel). The nuclei (red) were stained with DAPI. Note extensive transfer of gap junctional permeable tracer, Lucifer Yellow, in Cx32-expressing LNCaP clone (bottom right panel) and lack of transfer in control cells (top right panel). The microinjected cells are marked by the yellow arrows. For Western blot analysis, 5 μg of total protein was loaded in each lane. After analyzing the expression of Cx32, the blot was stripped and reprobed for β-actin to verify equal loading. The position of the molecular weight markers is indicated on the left.
Retroviral Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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plxsn retroviral vector  (TaKaRa)
95
TaKaRa plxsn retroviral vector
<t>Retroviral</t> expression of Cx32 and gap junctional communication in androgen-responsive LNCaP cells. LNCaP cells were infected with the control and Cx32-harboring recombinant retroviruses, and Cx32 expression level and junctional communication were determined by Western blot (A), immunocytochemical (B, left panels) and functional (B, right panels) analyses. (A) Note that only clones infected with Cx32-harboring retroviruses (lanes LN-32-1 and LN-32-2) express Cx32 (a 27-kDa monomer and a 54-kDa dimer), whereas a control clone infected with the control retrovirus (lane LN-NEO-1) and parental, early passage cells (lane LN-WT) do not. (B) Note that Cx32-expressing LNCaP cells form gap junctions (green, bottom left panel, yellow arrows), whereas no gap junctions are detected in control cells (top left panel). The nuclei (red) were stained with DAPI. Note extensive transfer of gap junctional permeable tracer, Lucifer Yellow, in Cx32-expressing LNCaP clone (bottom right panel) and lack of transfer in control cells (top right panel). The microinjected cells are marked by the yellow arrows. For Western blot analysis, 5 μg of total protein was loaded in each lane. After analyzing the expression of Cx32, the blot was stripped and reprobed for β-actin to verify equal loading. The position of the molecular weight markers is indicated on the left.
Plxsn Retroviral Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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lxin retroviral vector  (TaKaRa)
95
TaKaRa lxin retroviral vector
Drug selection eliminates DNA methylation of helper virus and vector from the VPC population. Genomic DNA extracted from AMIZ cells transfected with the LEIN vector with Zeocin and G418 selection or without drug selection on days 0 (lanes 3 and 4), 56 (lanes 5 to 8), and 67 (lanes 9 to 12) was first digested with DraI and EcoRV and divided into two equal portions. One portion was subjected to methylation-sensitive SmaI restriction endonuclease digestion, and the other was not. (A) Schema of the helper virus and vectors showing the locations of restriction enzyme sites and probes used for methylation analysis. D, DraI; E, EcoRV; S, SmaI; AAA, SV40 polyadenylation signal. Drawings are not to scale. (B) Hybridization of the gag DNA probe to detect helper virus 5′ LTR. SmaI digestion reduced the 1.8-kb band (even-numbered lanes from 4 to 12) to 1.5 kb (odd-numbered lanes from 3 to 11). DNA from NIH 3T3 cells was used to show the presence of endogenous <t>retroviral</t> elements (lanes 1 and 2). Since a 1.8-kb band was generated from the endogenous retroviral element after SmaI digestion (lane 1), the values for SmaI resistance were measured by densitometry as the relative intensities of the 1.8-kb bands without SmaI digestion and the 1.5-kb bands after SmaI digestion. (C) A green fluorescent protein DNA probe was used to detect the 5′ LTR of the LEIN vector. SmaI digestion reduced the 3.7-kb fragment to 3.4-, 2.7-, and 2.4-kb fragments, depending on the methylation status of the SmaI site in LEIN vector. (D) A 0.68-kb DNA fragment digested from the gag gene of pPAM3 by BstXI was used as a probe to detect a 1.2-kb band of the endogenous retroviral element to demonstrate relative loading in paired digestions with and without SmaI.
Lxin Retroviral Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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retrovirus vectors  (TaKaRa)
95
TaKaRa retrovirus vectors
Drug selection eliminates DNA methylation of helper virus and vector from the VPC population. Genomic DNA extracted from AMIZ cells transfected with the LEIN vector with Zeocin and G418 selection or without drug selection on days 0 (lanes 3 and 4), 56 (lanes 5 to 8), and 67 (lanes 9 to 12) was first digested with DraI and EcoRV and divided into two equal portions. One portion was subjected to methylation-sensitive SmaI restriction endonuclease digestion, and the other was not. (A) Schema of the helper virus and vectors showing the locations of restriction enzyme sites and probes used for methylation analysis. D, DraI; E, EcoRV; S, SmaI; AAA, SV40 polyadenylation signal. Drawings are not to scale. (B) Hybridization of the gag DNA probe to detect helper virus 5′ LTR. SmaI digestion reduced the 1.8-kb band (even-numbered lanes from 4 to 12) to 1.5 kb (odd-numbered lanes from 3 to 11). DNA from NIH 3T3 cells was used to show the presence of endogenous <t>retroviral</t> elements (lanes 1 and 2). Since a 1.8-kb band was generated from the endogenous retroviral element after SmaI digestion (lane 1), the values for SmaI resistance were measured by densitometry as the relative intensities of the 1.8-kb bands without SmaI digestion and the 1.5-kb bands after SmaI digestion. (C) A green fluorescent protein DNA probe was used to detect the 5′ LTR of the LEIN vector. SmaI digestion reduced the 3.7-kb fragment to 3.4-, 2.7-, and 2.4-kb fragments, depending on the methylation status of the SmaI site in LEIN vector. (D) A 0.68-kb DNA fragment digested from the gag gene of pPAM3 by BstXI was used as a probe to detect a 1.2-kb band of the endogenous retroviral element to demonstrate relative loading in paired digestions with and without SmaI.
Retrovirus Vectors, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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retroviral vector pqcxix  (TaKaRa)
95
TaKaRa retroviral vector pqcxix
Drug selection eliminates DNA methylation of helper virus and vector from the VPC population. Genomic DNA extracted from AMIZ cells transfected with the LEIN vector with Zeocin and G418 selection or without drug selection on days 0 (lanes 3 and 4), 56 (lanes 5 to 8), and 67 (lanes 9 to 12) was first digested with DraI and EcoRV and divided into two equal portions. One portion was subjected to methylation-sensitive SmaI restriction endonuclease digestion, and the other was not. (A) Schema of the helper virus and vectors showing the locations of restriction enzyme sites and probes used for methylation analysis. D, DraI; E, EcoRV; S, SmaI; AAA, SV40 polyadenylation signal. Drawings are not to scale. (B) Hybridization of the gag DNA probe to detect helper virus 5′ LTR. SmaI digestion reduced the 1.8-kb band (even-numbered lanes from 4 to 12) to 1.5 kb (odd-numbered lanes from 3 to 11). DNA from NIH 3T3 cells was used to show the presence of endogenous <t>retroviral</t> elements (lanes 1 and 2). Since a 1.8-kb band was generated from the endogenous retroviral element after SmaI digestion (lane 1), the values for SmaI resistance were measured by densitometry as the relative intensities of the 1.8-kb bands without SmaI digestion and the 1.5-kb bands after SmaI digestion. (C) A green fluorescent protein DNA probe was used to detect the 5′ LTR of the LEIN vector. SmaI digestion reduced the 3.7-kb fragment to 3.4-, 2.7-, and 2.4-kb fragments, depending on the methylation status of the SmaI site in LEIN vector. (D) A 0.68-kb DNA fragment digested from the gag gene of pPAM3 by BstXI was used as a probe to detect a 1.2-kb band of the endogenous retroviral element to demonstrate relative loading in paired digestions with and without SmaI.
Retroviral Vector Pqcxix, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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plhcx  (TaKaRa)
95
TaKaRa plhcx
Drug selection eliminates DNA methylation of helper virus and vector from the VPC population. Genomic DNA extracted from AMIZ cells transfected with the LEIN vector with Zeocin and G418 selection or without drug selection on days 0 (lanes 3 and 4), 56 (lanes 5 to 8), and 67 (lanes 9 to 12) was first digested with DraI and EcoRV and divided into two equal portions. One portion was subjected to methylation-sensitive SmaI restriction endonuclease digestion, and the other was not. (A) Schema of the helper virus and vectors showing the locations of restriction enzyme sites and probes used for methylation analysis. D, DraI; E, EcoRV; S, SmaI; AAA, SV40 polyadenylation signal. Drawings are not to scale. (B) Hybridization of the gag DNA probe to detect helper virus 5′ LTR. SmaI digestion reduced the 1.8-kb band (even-numbered lanes from 4 to 12) to 1.5 kb (odd-numbered lanes from 3 to 11). DNA from NIH 3T3 cells was used to show the presence of endogenous <t>retroviral</t> elements (lanes 1 and 2). Since a 1.8-kb band was generated from the endogenous retroviral element after SmaI digestion (lane 1), the values for SmaI resistance were measured by densitometry as the relative intensities of the 1.8-kb bands without SmaI digestion and the 1.5-kb bands after SmaI digestion. (C) A green fluorescent protein DNA probe was used to detect the 5′ LTR of the LEIN vector. SmaI digestion reduced the 3.7-kb fragment to 3.4-, 2.7-, and 2.4-kb fragments, depending on the methylation status of the SmaI site in LEIN vector. (D) A 0.68-kb DNA fragment digested from the gag gene of pPAM3 by BstXI was used as a probe to detect a 1.2-kb band of the endogenous retroviral element to demonstrate relative loading in paired digestions with and without SmaI.
Plhcx, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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liquid chromatography  (KCAS Bioanalytical and Biomarker Services)
93
KCAS Bioanalytical and Biomarker Services liquid chromatography
Drug selection eliminates DNA methylation of helper virus and vector from the VPC population. Genomic DNA extracted from AMIZ cells transfected with the LEIN vector with Zeocin and G418 selection or without drug selection on days 0 (lanes 3 and 4), 56 (lanes 5 to 8), and 67 (lanes 9 to 12) was first digested with DraI and EcoRV and divided into two equal portions. One portion was subjected to methylation-sensitive SmaI restriction endonuclease digestion, and the other was not. (A) Schema of the helper virus and vectors showing the locations of restriction enzyme sites and probes used for methylation analysis. D, DraI; E, EcoRV; S, SmaI; AAA, SV40 polyadenylation signal. Drawings are not to scale. (B) Hybridization of the gag DNA probe to detect helper virus 5′ LTR. SmaI digestion reduced the 1.8-kb band (even-numbered lanes from 4 to 12) to 1.5 kb (odd-numbered lanes from 3 to 11). DNA from NIH 3T3 cells was used to show the presence of endogenous <t>retroviral</t> elements (lanes 1 and 2). Since a 1.8-kb band was generated from the endogenous retroviral element after SmaI digestion (lane 1), the values for SmaI resistance were measured by densitometry as the relative intensities of the 1.8-kb bands without SmaI digestion and the 1.5-kb bands after SmaI digestion. (C) A green fluorescent protein DNA probe was used to detect the 5′ LTR of the LEIN vector. SmaI digestion reduced the 3.7-kb fragment to 3.4-, 2.7-, and 2.4-kb fragments, depending on the methylation status of the SmaI site in LEIN vector. (D) A 0.68-kb DNA fragment digested from the gag gene of pPAM3 by BstXI was used as a probe to detect a 1.2-kb band of the endogenous retroviral element to demonstrate relative loading in paired digestions with and without SmaI.
Liquid Chromatography, supplied by KCAS Bioanalytical and Biomarker Services, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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liquid chromatography - by Bioz Stars, 2026-03
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retroviral tet-off expression vectors  (TaKaRa)
94
TaKaRa retroviral tet-off expression vectors
Drug selection eliminates DNA methylation of helper virus and vector from the VPC population. Genomic DNA extracted from AMIZ cells transfected with the LEIN vector with Zeocin and G418 selection or without drug selection on days 0 (lanes 3 and 4), 56 (lanes 5 to 8), and 67 (lanes 9 to 12) was first digested with DraI and EcoRV and divided into two equal portions. One portion was subjected to methylation-sensitive SmaI restriction endonuclease digestion, and the other was not. (A) Schema of the helper virus and vectors showing the locations of restriction enzyme sites and probes used for methylation analysis. D, DraI; E, EcoRV; S, SmaI; AAA, SV40 polyadenylation signal. Drawings are not to scale. (B) Hybridization of the gag DNA probe to detect helper virus 5′ LTR. SmaI digestion reduced the 1.8-kb band (even-numbered lanes from 4 to 12) to 1.5 kb (odd-numbered lanes from 3 to 11). DNA from NIH 3T3 cells was used to show the presence of endogenous <t>retroviral</t> elements (lanes 1 and 2). Since a 1.8-kb band was generated from the endogenous retroviral element after SmaI digestion (lane 1), the values for SmaI resistance were measured by densitometry as the relative intensities of the 1.8-kb bands without SmaI digestion and the 1.5-kb bands after SmaI digestion. (C) A green fluorescent protein DNA probe was used to detect the 5′ LTR of the LEIN vector. SmaI digestion reduced the 3.7-kb fragment to 3.4-, 2.7-, and 2.4-kb fragments, depending on the methylation status of the SmaI site in LEIN vector. (D) A 0.68-kb DNA fragment digested from the gag gene of pPAM3 by BstXI was used as a probe to detect a 1.2-kb band of the endogenous retroviral element to demonstrate relative loading in paired digestions with and without SmaI.
Retroviral Tet Off Expression Vectors, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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kcas bio analytical  (KCAS Bioanalytical and Biomarker Services)
99
KCAS Bioanalytical and Biomarker Services kcas bio analytical
Drug selection eliminates DNA methylation of helper virus and vector from the VPC population. Genomic DNA extracted from AMIZ cells transfected with the LEIN vector with Zeocin and G418 selection or without drug selection on days 0 (lanes 3 and 4), 56 (lanes 5 to 8), and 67 (lanes 9 to 12) was first digested with DraI and EcoRV and divided into two equal portions. One portion was subjected to methylation-sensitive SmaI restriction endonuclease digestion, and the other was not. (A) Schema of the helper virus and vectors showing the locations of restriction enzyme sites and probes used for methylation analysis. D, DraI; E, EcoRV; S, SmaI; AAA, SV40 polyadenylation signal. Drawings are not to scale. (B) Hybridization of the gag DNA probe to detect helper virus 5′ LTR. SmaI digestion reduced the 1.8-kb band (even-numbered lanes from 4 to 12) to 1.5 kb (odd-numbered lanes from 3 to 11). DNA from NIH 3T3 cells was used to show the presence of endogenous <t>retroviral</t> elements (lanes 1 and 2). Since a 1.8-kb band was generated from the endogenous retroviral element after SmaI digestion (lane 1), the values for SmaI resistance were measured by densitometry as the relative intensities of the 1.8-kb bands without SmaI digestion and the 1.5-kb bands after SmaI digestion. (C) A green fluorescent protein DNA probe was used to detect the 5′ LTR of the LEIN vector. SmaI digestion reduced the 3.7-kb fragment to 3.4-, 2.7-, and 2.4-kb fragments, depending on the methylation status of the SmaI site in LEIN vector. (D) A 0.68-kb DNA fragment digested from the gag gene of pPAM3 by BstXI was used as a probe to detect a 1.2-kb band of the endogenous retroviral element to demonstrate relative loading in paired digestions with and without SmaI.
Kcas Bio Analytical, supplied by KCAS Bioanalytical and Biomarker Services, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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plxsn retroviral vector  (Becton Dickinson)
90
Becton Dickinson plxsn retroviral vector
Drug selection eliminates DNA methylation of helper virus and vector from the VPC population. Genomic DNA extracted from AMIZ cells transfected with the LEIN vector with Zeocin and G418 selection or without drug selection on days 0 (lanes 3 and 4), 56 (lanes 5 to 8), and 67 (lanes 9 to 12) was first digested with DraI and EcoRV and divided into two equal portions. One portion was subjected to methylation-sensitive SmaI restriction endonuclease digestion, and the other was not. (A) Schema of the helper virus and vectors showing the locations of restriction enzyme sites and probes used for methylation analysis. D, DraI; E, EcoRV; S, SmaI; AAA, SV40 polyadenylation signal. Drawings are not to scale. (B) Hybridization of the gag DNA probe to detect helper virus 5′ LTR. SmaI digestion reduced the 1.8-kb band (even-numbered lanes from 4 to 12) to 1.5 kb (odd-numbered lanes from 3 to 11). DNA from NIH 3T3 cells was used to show the presence of endogenous <t>retroviral</t> elements (lanes 1 and 2). Since a 1.8-kb band was generated from the endogenous retroviral element after SmaI digestion (lane 1), the values for SmaI resistance were measured by densitometry as the relative intensities of the 1.8-kb bands without SmaI digestion and the 1.5-kb bands after SmaI digestion. (C) A green fluorescent protein DNA probe was used to detect the 5′ LTR of the LEIN vector. SmaI digestion reduced the 3.7-kb fragment to 3.4-, 2.7-, and 2.4-kb fragments, depending on the methylation status of the SmaI site in LEIN vector. (D) A 0.68-kb DNA fragment digested from the gag gene of pPAM3 by BstXI was used as a probe to detect a 1.2-kb band of the endogenous retroviral element to demonstrate relative loading in paired digestions with and without SmaI.
Plxsn Retroviral Vector, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pvsv-g retroviral vector  (Becton Dickinson)
90
Becton Dickinson pvsv-g retroviral vector
Drug selection eliminates DNA methylation of helper virus and vector from the VPC population. Genomic DNA extracted from AMIZ cells transfected with the LEIN vector with Zeocin and G418 selection or without drug selection on days 0 (lanes 3 and 4), 56 (lanes 5 to 8), and 67 (lanes 9 to 12) was first digested with DraI and EcoRV and divided into two equal portions. One portion was subjected to methylation-sensitive SmaI restriction endonuclease digestion, and the other was not. (A) Schema of the helper virus and vectors showing the locations of restriction enzyme sites and probes used for methylation analysis. D, DraI; E, EcoRV; S, SmaI; AAA, SV40 polyadenylation signal. Drawings are not to scale. (B) Hybridization of the gag DNA probe to detect helper virus 5′ LTR. SmaI digestion reduced the 1.8-kb band (even-numbered lanes from 4 to 12) to 1.5 kb (odd-numbered lanes from 3 to 11). DNA from NIH 3T3 cells was used to show the presence of endogenous <t>retroviral</t> elements (lanes 1 and 2). Since a 1.8-kb band was generated from the endogenous retroviral element after SmaI digestion (lane 1), the values for SmaI resistance were measured by densitometry as the relative intensities of the 1.8-kb bands without SmaI digestion and the 1.5-kb bands after SmaI digestion. (C) A green fluorescent protein DNA probe was used to detect the 5′ LTR of the LEIN vector. SmaI digestion reduced the 3.7-kb fragment to 3.4-, 2.7-, and 2.4-kb fragments, depending on the methylation status of the SmaI site in LEIN vector. (D) A 0.68-kb DNA fragment digested from the gag gene of pPAM3 by BstXI was used as a probe to detect a 1.2-kb band of the endogenous retroviral element to demonstrate relative loading in paired digestions with and without SmaI.
Pvsv G Retroviral Vector, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Retroviral expression of Cx32 and gap junctional communication in androgen-responsive LNCaP cells. LNCaP cells were infected with the control and Cx32-harboring recombinant retroviruses, and Cx32 expression level and junctional communication were determined by Western blot (A), immunocytochemical (B, left panels) and functional (B, right panels) analyses. (A) Note that only clones infected with Cx32-harboring retroviruses (lanes LN-32-1 and LN-32-2) express Cx32 (a 27-kDa monomer and a 54-kDa dimer), whereas a control clone infected with the control retrovirus (lane LN-NEO-1) and parental, early passage cells (lane LN-WT) do not. (B) Note that Cx32-expressing LNCaP cells form gap junctions (green, bottom left panel, yellow arrows), whereas no gap junctions are detected in control cells (top left panel). The nuclei (red) were stained with DAPI. Note extensive transfer of gap junctional permeable tracer, Lucifer Yellow, in Cx32-expressing LNCaP clone (bottom right panel) and lack of transfer in control cells (top right panel). The microinjected cells are marked by the yellow arrows. For Western blot analysis, 5 μg of total protein was loaded in each lane. After analyzing the expression of Cx32, the blot was stripped and reprobed for β-actin to verify equal loading. The position of the molecular weight markers is indicated on the left.

Journal:

Article Title: Androgen-regulated Formation and Degradation of Gap Junctions in Androgen-responsive Human Prostate Cancer Cells

doi: 10.1091/mbc.E06-04-0280

Figure Lengend Snippet: Retroviral expression of Cx32 and gap junctional communication in androgen-responsive LNCaP cells. LNCaP cells were infected with the control and Cx32-harboring recombinant retroviruses, and Cx32 expression level and junctional communication were determined by Western blot (A), immunocytochemical (B, left panels) and functional (B, right panels) analyses. (A) Note that only clones infected with Cx32-harboring retroviruses (lanes LN-32-1 and LN-32-2) express Cx32 (a 27-kDa monomer and a 54-kDa dimer), whereas a control clone infected with the control retrovirus (lane LN-NEO-1) and parental, early passage cells (lane LN-WT) do not. (B) Note that Cx32-expressing LNCaP cells form gap junctions (green, bottom left panel, yellow arrows), whereas no gap junctions are detected in control cells (top left panel). The nuclei (red) were stained with DAPI. Note extensive transfer of gap junctional permeable tracer, Lucifer Yellow, in Cx32-expressing LNCaP clone (bottom right panel) and lack of transfer in control cells (top right panel). The microinjected cells are marked by the yellow arrows. For Western blot analysis, 5 μg of total protein was loaded in each lane. After analyzing the expression of Cx32, the blot was stripped and reprobed for β-actin to verify equal loading. The position of the molecular weight markers is indicated on the left.

Article Snippet: A full-length AR cDNA (a kind gift from Dr. K. Burnstein, University of Miami School of Medicine) was subcloned into EcoRI site of retroviral vector, pMSCV-puro (Clontech Laboratories).

Techniques: Retroviral, Expressing, Infection, Control, Recombinant, Western Blot, Functional Assay, Clone Assay, Staining, Molecular Weight

Drug selection eliminates DNA methylation of helper virus and vector from the VPC population. Genomic DNA extracted from AMIZ cells transfected with the LEIN vector with Zeocin and G418 selection or without drug selection on days 0 (lanes 3 and 4), 56 (lanes 5 to 8), and 67 (lanes 9 to 12) was first digested with DraI and EcoRV and divided into two equal portions. One portion was subjected to methylation-sensitive SmaI restriction endonuclease digestion, and the other was not. (A) Schema of the helper virus and vectors showing the locations of restriction enzyme sites and probes used for methylation analysis. D, DraI; E, EcoRV; S, SmaI; AAA, SV40 polyadenylation signal. Drawings are not to scale. (B) Hybridization of the gag DNA probe to detect helper virus 5′ LTR. SmaI digestion reduced the 1.8-kb band (even-numbered lanes from 4 to 12) to 1.5 kb (odd-numbered lanes from 3 to 11). DNA from NIH 3T3 cells was used to show the presence of endogenous retroviral elements (lanes 1 and 2). Since a 1.8-kb band was generated from the endogenous retroviral element after SmaI digestion (lane 1), the values for SmaI resistance were measured by densitometry as the relative intensities of the 1.8-kb bands without SmaI digestion and the 1.5-kb bands after SmaI digestion. (C) A green fluorescent protein DNA probe was used to detect the 5′ LTR of the LEIN vector. SmaI digestion reduced the 3.7-kb fragment to 3.4-, 2.7-, and 2.4-kb fragments, depending on the methylation status of the SmaI site in LEIN vector. (D) A 0.68-kb DNA fragment digested from the gag gene of pPAM3 by BstXI was used as a probe to detect a 1.2-kb band of the endogenous retroviral element to demonstrate relative loading in paired digestions with and without SmaI.

Journal:

Article Title: Chimeric Retroviral Helper Virus and Picornavirus IRES Sequence To Eliminate DNA Methylation for Improved Retroviral Packaging Cells

doi:

Figure Lengend Snippet: Drug selection eliminates DNA methylation of helper virus and vector from the VPC population. Genomic DNA extracted from AMIZ cells transfected with the LEIN vector with Zeocin and G418 selection or without drug selection on days 0 (lanes 3 and 4), 56 (lanes 5 to 8), and 67 (lanes 9 to 12) was first digested with DraI and EcoRV and divided into two equal portions. One portion was subjected to methylation-sensitive SmaI restriction endonuclease digestion, and the other was not. (A) Schema of the helper virus and vectors showing the locations of restriction enzyme sites and probes used for methylation analysis. D, DraI; E, EcoRV; S, SmaI; AAA, SV40 polyadenylation signal. Drawings are not to scale. (B) Hybridization of the gag DNA probe to detect helper virus 5′ LTR. SmaI digestion reduced the 1.8-kb band (even-numbered lanes from 4 to 12) to 1.5 kb (odd-numbered lanes from 3 to 11). DNA from NIH 3T3 cells was used to show the presence of endogenous retroviral elements (lanes 1 and 2). Since a 1.8-kb band was generated from the endogenous retroviral element after SmaI digestion (lane 1), the values for SmaI resistance were measured by densitometry as the relative intensities of the 1.8-kb bands without SmaI digestion and the 1.5-kb bands after SmaI digestion. (C) A green fluorescent protein DNA probe was used to detect the 5′ LTR of the LEIN vector. SmaI digestion reduced the 3.7-kb fragment to 3.4-, 2.7-, and 2.4-kb fragments, depending on the methylation status of the SmaI site in LEIN vector. (D) A 0.68-kb DNA fragment digested from the gag gene of pPAM3 by BstXI was used as a probe to detect a 1.2-kb band of the endogenous retroviral element to demonstrate relative loading in paired digestions with and without SmaI.

Article Snippet: An IRES sequence of encephalomyocarditis virus was isolated from the LXIN retroviral vector (Clontech, Palo Alto, Calif.) by Nsi I and Pst I digestions and inserted into a Pst I-linearized pZeoSV mammalian expression vector (Invitrogen, Carlsbad, Calif.) immediately 5′ of the EM-7 prokaryotic promoter/Zeocin resistance gene (Zeo) to create an IRES-Zeo expression cassette in plasmid pIRES-Zeo-SV40.

Techniques: Selection, DNA Methylation Assay, Plasmid Preparation, Transfection, Methylation, Hybridization, Generated