Journal: Current protocols in microbiology

Article Title: Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes

doi: 10.1002/cpmc.101

Figure Lengend Snippet: A. qPCR was performed to measure HPV18 DNA in fractions prepared by standard (Basic Protocol 1) and Ripcord (Alternative Protocol 2).

Article Snippet: Materials list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 HPV genome such as pUC-HPV18 ( Cole & Danos, 1987 ) or pBR-HPV18 ( Boshart et al., 1984 ) (HPV genomes can be acquired from The HPV Reference Center) NcoI (50,000 U/ml) (NEB #R0193T), EcoRI (20,000 U/ml) (NEB# R0101L) or other enzyme to release viral genome from the bacterial vector NEB CutSmart Buffer (NEB #B7204S) QIAquick PCR Purification Kit (Qiagen #28104) Gel Loading Dye, Purple (6X) (NEB #B7024S) SeaKem GTG Agarose (Lonza #50074) Ethidium Bromide (Invitrogen #15585011) TAE Buffer (Tris-acetate-EDTA) (50X) (Thermo Fisher #B49) T4 DNA ligase (400,000 U/ml) (NEB #M0202S) 10X T4 DNA Ligase Reaction Buffer (NEB #B0202S) Isopropanol (Sigma #190764) 5M NaCl (KD Medical #RGF-3270) 70% Ethanol TE pH 8.0 (Quality Biological #351-011-131) Heat block capable of cooling such as ThermoMixer C (Eppendorf #5382000023) or 16°C water bath in a cold room Refrigerated tabletop centrifuge such as Centrifuge 5415R (Eppendorf #5401000137) Agarose gel running apparatus list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Prepare a 50 μl restriction digestion reaction to free HPV18 from the bacterial plasmid backbone containing 10 μg pUC-HPV18, 1X CutSmart Buffer, and 50 U NcoI.

Techniques:

Journal: Current protocols in microbiology

Article Title: Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes

doi: 10.1002/cpmc.101

Figure Lengend Snippet: Quasivirus stocks were digested with nuclease to remove DNA outside of the capsid and the viral DNA was extracted and quantified by qPCR for HPV18 DNA. Shown are examples of viral genome equivalents (VGE) per μl obtained from quasivirus stocks prepared with Basic Protocol 1: Transfection, Harvest, and Isolation of HPV Quasiviruses; Alternate Protocol 1: Packaging HPV DNA Replicated in 293TT Cells; Support Protocol 1: Production of HPV Minicircles and Alternate Support Protocol 1: Production of Recircularized HPV Genomes.

Article Snippet: Materials list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 HPV genome such as pUC-HPV18 ( Cole & Danos, 1987 ) or pBR-HPV18 ( Boshart et al., 1984 ) (HPV genomes can be acquired from The HPV Reference Center) NcoI (50,000 U/ml) (NEB #R0193T), EcoRI (20,000 U/ml) (NEB# R0101L) or other enzyme to release viral genome from the bacterial vector NEB CutSmart Buffer (NEB #B7204S) QIAquick PCR Purification Kit (Qiagen #28104) Gel Loading Dye, Purple (6X) (NEB #B7024S) SeaKem GTG Agarose (Lonza #50074) Ethidium Bromide (Invitrogen #15585011) TAE Buffer (Tris-acetate-EDTA) (50X) (Thermo Fisher #B49) T4 DNA ligase (400,000 U/ml) (NEB #M0202S) 10X T4 DNA Ligase Reaction Buffer (NEB #B0202S) Isopropanol (Sigma #190764) 5M NaCl (KD Medical #RGF-3270) 70% Ethanol TE pH 8.0 (Quality Biological #351-011-131) Heat block capable of cooling such as ThermoMixer C (Eppendorf #5382000023) or 16°C water bath in a cold room Refrigerated tabletop centrifuge such as Centrifuge 5415R (Eppendorf #5401000137) Agarose gel running apparatus list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Prepare a 50 μl restriction digestion reaction to free HPV18 from the bacterial plasmid backbone containing 10 μg pUC-HPV18, 1X CutSmart Buffer, and 50 U NcoI.

Techniques: Transfection, Isolation

Journal: Current protocols in microbiology

Article Title: Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes

doi: 10.1002/cpmc.101

Figure Lengend Snippet: A. Map of pMC.BESPX-HPV18

Article Snippet: Materials list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 HPV genome such as pUC-HPV18 ( Cole & Danos, 1987 ) or pBR-HPV18 ( Boshart et al., 1984 ) (HPV genomes can be acquired from The HPV Reference Center) NcoI (50,000 U/ml) (NEB #R0193T), EcoRI (20,000 U/ml) (NEB# R0101L) or other enzyme to release viral genome from the bacterial vector NEB CutSmart Buffer (NEB #B7204S) QIAquick PCR Purification Kit (Qiagen #28104) Gel Loading Dye, Purple (6X) (NEB #B7024S) SeaKem GTG Agarose (Lonza #50074) Ethidium Bromide (Invitrogen #15585011) TAE Buffer (Tris-acetate-EDTA) (50X) (Thermo Fisher #B49) T4 DNA ligase (400,000 U/ml) (NEB #M0202S) 10X T4 DNA Ligase Reaction Buffer (NEB #B0202S) Isopropanol (Sigma #190764) 5M NaCl (KD Medical #RGF-3270) 70% Ethanol TE pH 8.0 (Quality Biological #351-011-131) Heat block capable of cooling such as ThermoMixer C (Eppendorf #5382000023) or 16°C water bath in a cold room Refrigerated tabletop centrifuge such as Centrifuge 5415R (Eppendorf #5401000137) Agarose gel running apparatus list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Prepare a 50 μl restriction digestion reaction to free HPV18 from the bacterial plasmid backbone containing 10 μg pUC-HPV18, 1X CutSmart Buffer, and 50 U NcoI.

Techniques:

Journal: Current protocols in microbiology

Article Title: Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes

doi: 10.1002/cpmc.101

Figure Lengend Snippet: A. Map of pUC-HPV18 showing the NcoI sites used to remove the vector and recircularize the viral genome.

Article Snippet: Materials list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 HPV genome such as pUC-HPV18 ( Cole & Danos, 1987 ) or pBR-HPV18 ( Boshart et al., 1984 ) (HPV genomes can be acquired from The HPV Reference Center) NcoI (50,000 U/ml) (NEB #R0193T), EcoRI (20,000 U/ml) (NEB# R0101L) or other enzyme to release viral genome from the bacterial vector NEB CutSmart Buffer (NEB #B7204S) QIAquick PCR Purification Kit (Qiagen #28104) Gel Loading Dye, Purple (6X) (NEB #B7024S) SeaKem GTG Agarose (Lonza #50074) Ethidium Bromide (Invitrogen #15585011) TAE Buffer (Tris-acetate-EDTA) (50X) (Thermo Fisher #B49) T4 DNA ligase (400,000 U/ml) (NEB #M0202S) 10X T4 DNA Ligase Reaction Buffer (NEB #B0202S) Isopropanol (Sigma #190764) 5M NaCl (KD Medical #RGF-3270) 70% Ethanol TE pH 8.0 (Quality Biological #351-011-131) Heat block capable of cooling such as ThermoMixer C (Eppendorf #5382000023) or 16°C water bath in a cold room Refrigerated tabletop centrifuge such as Centrifuge 5415R (Eppendorf #5401000137) Agarose gel running apparatus list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Prepare a 50 μl restriction digestion reaction to free HPV18 from the bacterial plasmid backbone containing 10 μg pUC-HPV18, 1X CutSmart Buffer, and 50 U NcoI.

Techniques: Plasmid Preparation

Journal: Current protocols in microbiology

Article Title: Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes

doi: 10.1002/cpmc.101

Figure Lengend Snippet: A. Duplicate wells of HFKs were infected with HPV18 Quasivirus (prepared by Basic Protocol 1) at an MOI of 100. RNA was collected at 24, 48, 72, and 96 hpi. RT-qPCR for viral transcripts E1Ê4 (left) and E6*I (right) was performed to quantitate viral transcription. Error bars represent the standard deviation of replicate wells.

Article Snippet: Materials list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 HPV genome such as pUC-HPV18 ( Cole & Danos, 1987 ) or pBR-HPV18 ( Boshart et al., 1984 ) (HPV genomes can be acquired from The HPV Reference Center) NcoI (50,000 U/ml) (NEB #R0193T), EcoRI (20,000 U/ml) (NEB# R0101L) or other enzyme to release viral genome from the bacterial vector NEB CutSmart Buffer (NEB #B7204S) QIAquick PCR Purification Kit (Qiagen #28104) Gel Loading Dye, Purple (6X) (NEB #B7024S) SeaKem GTG Agarose (Lonza #50074) Ethidium Bromide (Invitrogen #15585011) TAE Buffer (Tris-acetate-EDTA) (50X) (Thermo Fisher #B49) T4 DNA ligase (400,000 U/ml) (NEB #M0202S) 10X T4 DNA Ligase Reaction Buffer (NEB #B0202S) Isopropanol (Sigma #190764) 5M NaCl (KD Medical #RGF-3270) 70% Ethanol TE pH 8.0 (Quality Biological #351-011-131) Heat block capable of cooling such as ThermoMixer C (Eppendorf #5382000023) or 16°C water bath in a cold room Refrigerated tabletop centrifuge such as Centrifuge 5415R (Eppendorf #5401000137) Agarose gel running apparatus list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Prepare a 50 μl restriction digestion reaction to free HPV18 from the bacterial plasmid backbone containing 10 μg pUC-HPV18, 1X CutSmart Buffer, and 50 U NcoI.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation

Journal: Cancer gene therapy

Article Title: Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA.

doi: 10.1038/sj.cgt.7700891

Figure Lengend Snippet: Figure 1 Suppression of HPV18 E6 transient expression by siRNA. COS-1 cells were transfected with a FLAG-tagged HPV18 E6 expression plasmid along with the indicated siRNA for 20 h and then analyzed by immunoblotting. The level of E6 was quantified by densitometry and normalized to that of a-vimentin and then compared to cells transfected with the control siRNA.

Article Snippet: Cell lines and plasmid transfection HPV18 genome positive cell lines (HeLa, SW756) were purchased from American Type Culture Collection (ATCC, Rockville, MD), and COS-1 cells were provided by Japanese Collection of Research Biosources (JCRB).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Cancer gene therapy

Article Title: Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA.

doi: 10.1038/sj.cgt.7700891

Figure Lengend Snippet: Figure 2 Downregulation of HPV18 E6 and E7 mRNA expression in HeLa cells by siRNAs. HeLa cells were transfected with the indicated siRNA. After 3 days of incubation, cytoplasmic RNA was extracted and reverse-transcribed. cDNA samples were then serially diluted (1:1, 1:5, 1:25) and subjected to PCR. PCR products were separated on 2.5% agarose gels, stained with SYBR GOLD, and photographed. Intensities of the E6 and E7 bands from E6 siRNA- treated cells (1:1) were quantified by densitometry and normalized to that of the b-actin band. Relative E6 and E7 mRNA levels of E6 siRNA-treated cells (1:1) are shown. Buffer, buffer alone; Control, control siRNA; Sp-18E6, Sp-18E6 siRNA; Ex-18E6, Ex-18E6 siRNA.

Article Snippet: Cell lines and plasmid transfection HPV18 genome positive cell lines (HeLa, SW756) were purchased from American Type Culture Collection (ATCC, Rockville, MD), and COS-1 cells were provided by Japanese Collection of Research Biosources (JCRB).

Techniques: Expressing, Transfection, Incubation, Reverse Transcription, Staining, Control

Journal: Cancer gene therapy

Article Title: Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA.

doi: 10.1038/sj.cgt.7700891

Figure Lengend Snippet: Figure 4 Suppression of in vitro growth of HPV18-positive cells by siRNAs. Effects of siRNA on monolayer growth (a, b) and colony formation in soft agar cultures (c, d) of HeLa and SW756 cells. Cells were transfected with the indicated siRNA for 24 h, then trypsinized, and dispensed into a 96-well plate at 750 cells/well, after which cell viability was determined at 5 days after plating. Relative cell growth is shown. For colony formation assay, cells were retransfected with each siRNA for 24 h, then trypsinized and resuspended in complete medium containing 0.3% agar. A total of 600 cells were overlaid on medium containing 0.5% agar in a 12-well plate. The number of colonies consisting of more than 50 cells was determined 12 days after plating. Buffer, buffer alone; Control, control siRNA; Sp-18E6, Sp-18E6 siRNA; Ex-18E6, Ex-18E6 siRNA.

Article Snippet: Cell lines and plasmid transfection HPV18 genome positive cell lines (HeLa, SW756) were purchased from American Type Culture Collection (ATCC, Rockville, MD), and COS-1 cells were provided by Japanese Collection of Research Biosources (JCRB).

Techniques: In Vitro, Transfection, Colony Assay, Control

Journal: Current protocols in microbiology

Article Title: Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes.

doi: 10.1002/cpmc.101

Figure Lengend Snippet: Figure 4 Production of HPV18 minicircles. (A) Map of pMC.BESPX-HPV18. (B) Agarose gel showing pMC.BESPX-HPV18 DNA before and after induction. E. coli ZYCY10P3S2T transformed with pMC.BESPX- HPV18 were induced to generate minicircles.Plasmid DNA isolated before and after induction was digested with BglII. DNAs were separated on a 0.8% TAE agarose gel and imaged on a UV lightbox. Lane 1: Pre-induction pMC.BESPX-HPV18, undigested; lane 2: pre-induction pMC.BESPX-HPV18, digested; lane 3: post-induction pMC.BESPX-HPV18, undigested;lane 4:post-induction pMC.BESPX-HPV18, digested.CCC, covalently closed circular DNA.

Article Snippet: Materials 293TT cells (available from NCI’s Developmental Therapeutics Program) 293TT medium (see recipe) with and without antibiotics Opti-MEM serum-free medium (Thermo Fisher #31985070) Lipofectamine 2000 (Invitrogen #11668019) HPV genomes prepared using Support Protocol 1 or Support Protocol 2 using either of these plasmids: Plasmid pMC.BESPX-HPV18 minicircle HPV18 (Support Protocol 1; Henno et al., 2017) Recircularized HPV18 genomes (Support Protocol 2; Boshart et al., 1984; Cole & Danos, 1987) derived from cloned HPV genomes; HPV genomes can be requested from the HPV Reference Center p16SheLL (Addgene #37320) or p18SheLL (Addgene #37321) 0.25% trypsin/EDTA (Thermo Fisher #25200056) Dulbecco’s phosphate-buffered saline (DPBS with calcium and magnesium; Invitrogen #14040-141) MgCl2 (Quality Biological #351-033-721) Triton X-100 (Sigma #T8787) 1 M ammonium sulfate, pH 9 (Sigma #204501) Plasmid Safe (Lucigen #E3110K) Benzonase (Millipore #70664-3) 5 M NaCl (Sigma #59222C) DPBS/NaCl (see recipe) 46% OptiPrep solution (see recipe) 75-cm2 (T-75) tissue culture flasks (Cellstar #658175) 5% and 10% CO2 incubators, e.g., Heracell VIOS 160i (Thermo Fisher # 51030400) Serological pipettes 5-ml polypropylene round-bottom tubes (Corning #352063) 50-ml conical tubes (Corning #352070) Centrifuge such as Sorvall Legend XTR Centrifuge (Thermo Fisher #75004521) Refrigerated tabletop centrifuge such as Centrifuge 5415R (Eppendorf #5401000137) Siliconized conical microcentrifuge tubes with screw caps (Bio Plas #4202SLS) Heat block such as ThermoMixer C (Eppendorf #5382000023) 5-ml open-top thinwall polypropylene ultracentrifuge tubes (Beckman #326819) Porter and McBride 3 of 32 Current Protocols in Microbiology 3-ml syringe (VWR #53548-017) 150-mm stainless steel needle (Kemtech #S389150) Ultracentrifuge, such as OPTIMA XPN 90 (Beckman # A94468) Clamp stand 22-G needles (Monoject #15141-136) Part 1: Transfection of 293TT cells 1.

Techniques: Agarose Gel Electrophoresis, Transformation Assay, Plasmid Preparation, Isolation

Journal: Current protocols in microbiology

Article Title: Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes.

doi: 10.1002/cpmc.101

Figure Lengend Snippet: Figure 5 Traditional HPV genome recircularization. (A) Map of pUC-HPV18 showing the NcoI sites used to remove the vector and recircularize the viral genome. (B) Agarose gel showing a comparison of uncut, digested, and religated viral DNA. Lane 1: uncut pBR-HPV18; Lane 2, pBR-HPV18 restriction digested to free the HPV genome from plasmid backbone; Lane 3: pBR-HPV18 fragments ligated under dilute conditions to promote intramolecular recombination. Samples were separated on a 0.8% agarose TAE gel containing 0.5 μg/ml EtBr and imaged on a UV imager.

Article Snippet: Materials 293TT cells (available from NCI’s Developmental Therapeutics Program) 293TT medium (see recipe) with and without antibiotics Opti-MEM serum-free medium (Thermo Fisher #31985070) Lipofectamine 2000 (Invitrogen #11668019) HPV genomes prepared using Support Protocol 1 or Support Protocol 2 using either of these plasmids: Plasmid pMC.BESPX-HPV18 minicircle HPV18 (Support Protocol 1; Henno et al., 2017) Recircularized HPV18 genomes (Support Protocol 2; Boshart et al., 1984; Cole & Danos, 1987) derived from cloned HPV genomes; HPV genomes can be requested from the HPV Reference Center p16SheLL (Addgene #37320) or p18SheLL (Addgene #37321) 0.25% trypsin/EDTA (Thermo Fisher #25200056) Dulbecco’s phosphate-buffered saline (DPBS with calcium and magnesium; Invitrogen #14040-141) MgCl2 (Quality Biological #351-033-721) Triton X-100 (Sigma #T8787) 1 M ammonium sulfate, pH 9 (Sigma #204501) Plasmid Safe (Lucigen #E3110K) Benzonase (Millipore #70664-3) 5 M NaCl (Sigma #59222C) DPBS/NaCl (see recipe) 46% OptiPrep solution (see recipe) 75-cm2 (T-75) tissue culture flasks (Cellstar #658175) 5% and 10% CO2 incubators, e.g., Heracell VIOS 160i (Thermo Fisher # 51030400) Serological pipettes 5-ml polypropylene round-bottom tubes (Corning #352063) 50-ml conical tubes (Corning #352070) Centrifuge such as Sorvall Legend XTR Centrifuge (Thermo Fisher #75004521) Refrigerated tabletop centrifuge such as Centrifuge 5415R (Eppendorf #5401000137) Siliconized conical microcentrifuge tubes with screw caps (Bio Plas #4202SLS) Heat block such as ThermoMixer C (Eppendorf #5382000023) 5-ml open-top thinwall polypropylene ultracentrifuge tubes (Beckman #326819) Porter and McBride 3 of 32 Current Protocols in Microbiology 3-ml syringe (VWR #53548-017) 150-mm stainless steel needle (Kemtech #S389150) Ultracentrifuge, such as OPTIMA XPN 90 (Beckman # A94468) Clamp stand 22-G needles (Monoject #15141-136) Part 1: Transfection of 293TT cells 1.

Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Comparison

Journal: Current protocols in microbiology

Article Title: Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes.

doi: 10.1002/cpmc.101

Figure Lengend Snippet: Figure 6 Screening of quasivirus fractions. (A) qPCR was performed to measure HPV18 DNA in fractions prepared by standard (Basic Protocol 1) and Ripcord (Alternate Protocol 2). (B) Total proteins in each quasivirus fractions prepared by normal (Basic Protocol 1) and Ripcord (Alternate Protocol 2) methods were analyzed by SDS-PAGE and SYPRO Ruby detection.

Article Snippet: Materials 293TT cells (available from NCI’s Developmental Therapeutics Program) 293TT medium (see recipe) with and without antibiotics Opti-MEM serum-free medium (Thermo Fisher #31985070) Lipofectamine 2000 (Invitrogen #11668019) HPV genomes prepared using Support Protocol 1 or Support Protocol 2 using either of these plasmids: Plasmid pMC.BESPX-HPV18 minicircle HPV18 (Support Protocol 1; Henno et al., 2017) Recircularized HPV18 genomes (Support Protocol 2; Boshart et al., 1984; Cole & Danos, 1987) derived from cloned HPV genomes; HPV genomes can be requested from the HPV Reference Center p16SheLL (Addgene #37320) or p18SheLL (Addgene #37321) 0.25% trypsin/EDTA (Thermo Fisher #25200056) Dulbecco’s phosphate-buffered saline (DPBS with calcium and magnesium; Invitrogen #14040-141) MgCl2 (Quality Biological #351-033-721) Triton X-100 (Sigma #T8787) 1 M ammonium sulfate, pH 9 (Sigma #204501) Plasmid Safe (Lucigen #E3110K) Benzonase (Millipore #70664-3) 5 M NaCl (Sigma #59222C) DPBS/NaCl (see recipe) 46% OptiPrep solution (see recipe) 75-cm2 (T-75) tissue culture flasks (Cellstar #658175) 5% and 10% CO2 incubators, e.g., Heracell VIOS 160i (Thermo Fisher # 51030400) Serological pipettes 5-ml polypropylene round-bottom tubes (Corning #352063) 50-ml conical tubes (Corning #352070) Centrifuge such as Sorvall Legend XTR Centrifuge (Thermo Fisher #75004521) Refrigerated tabletop centrifuge such as Centrifuge 5415R (Eppendorf #5401000137) Siliconized conical microcentrifuge tubes with screw caps (Bio Plas #4202SLS) Heat block such as ThermoMixer C (Eppendorf #5382000023) 5-ml open-top thinwall polypropylene ultracentrifuge tubes (Beckman #326819) Porter and McBride 3 of 32 Current Protocols in Microbiology 3-ml syringe (VWR #53548-017) 150-mm stainless steel needle (Kemtech #S389150) Ultracentrifuge, such as OPTIMA XPN 90 (Beckman # A94468) Clamp stand 22-G needles (Monoject #15141-136) Part 1: Transfection of 293TT cells 1.

Techniques: SDS Page