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: Scientific Reports

Article Title: Novel recombinant papillomavirus genomes expressing selectable genes

doi: 10.1038/srep37782

Figure Lengend Snippet: ( a ) Recombinant HPV18-based genomes were generated to contain expression cassettes for selectable marker genes. A 1479 bp portion of the HPV18 L2 and L1 genes was replaced by the expression cassettes as described in Methods. ( b ) DNA was harvested from HFKs 96 hours post-electroporation. Prior to analysis, total cellular DNA was digested with NcoI and DpnI to linearize the genome and remove unreplicated input DNA. Viral DNA was detected by DNA qPCR. Data shown was normalized by cellular β-actin levels. The data are the averages of two independent experiments, each with three different isolates of keratinocytes. Data is grouped by experiment (left; n = 2), by isolate (middle; n = 3), and compiled (right; n = 6). Error bars indicate the standard error of the mean (SEM). ( c ) Cellular DNA was analyzed using Southern blot with a [ 32 P]-labeled HPV18 DNA probe. ( d ) Total cellular RNA was harvested from cells 96 hours post electroporation. Viral HPV18 E1^E4 and E6*I cDNA was quantified using qPCR. Data shown was normalized by cellular TBP levels. The data are the averages of two independent experiments, each with three different isolates of keratinocytes. Data is grouped by experiment (top; n = 2), by donor (middle; n = 3), and compiled (bottom; n = 6). Error bars indicate the standard error of the mean (SEM).

Article Snippet: To generate HPV18 genomes containing a GFP-Zeocin cassette, the GFP-Zeocin fusion gene was PCR amplified from pSELECT-CGFP (Invivogen; cat. no. psetz-zgfpsh) incorporating flanking BglII and BclI restriction sites.

Techniques: Recombinant, Generated, Expressing, Marker, Electroporation, Southern Blot, Labeling

Journal: Scientific Reports

Article Title: Novel recombinant papillomavirus genomes expressing selectable genes

doi: 10.1038/srep37782

Figure Lengend Snippet: 1 × 10 6 cells were electroporated with 1 μg of each indicated plasmid. 2 × 10 5 cells were plated on 10 cm plates. One-day post-electroporation cells were treated with G418 as indicated. Cells were cultured for approximately 2 weeks. ( A ) Keratinocyte colonies were stained with methylene blue. ( B ) DNA was harvested from colonies obtained following continuous selection. Viral DNA was detected by Southern blot analysis with a [ 32 P]-labeled HPV18 DNA probe.

Article Snippet: To generate HPV18 genomes containing a GFP-Zeocin cassette, the GFP-Zeocin fusion gene was PCR amplified from pSELECT-CGFP (Invivogen; cat. no. psetz-zgfpsh) incorporating flanking BglII and BclI restriction sites.

Techniques: Plasmid Preparation, Electroporation, Cell Culture, Staining, Selection, Southern Blot, Labeling

Journal: Scientific Reports

Article Title: Novel recombinant papillomavirus genomes expressing selectable genes

doi: 10.1038/srep37782

Figure Lengend Snippet: ( a ) Primary foreskin keratinocytes were electroporated with 2 μg HPV18-GFP::SH. 85,000 cells were plated in a well of a 12 well plate. 24 hours post transfection, cells were selected with 25 μg/ml zeocin. Selection was maintained for 96 hours, after which the selection was lowered to 2.5 μg/ml. An Incucyte live cell imager was used to image the entire well. Numbers indicate days post transfection. The scale bar equals 1.3 mm. ( b ) Primary foreskin keratinocytes were transfected with 2 μg HPV18-GFP::SH. 1 × 10 6 cells were plated on a 10 cm cell culture plate and selected as in ( a ). Colonies were pooled onto coverslips. Cells were fixed and GFP levels were detected using standard immunofluorescence methods. The image shows a keratinocyte colony surrounded by irradiated fibroblasts. The scale bar equals 25 μm. ( c ) Viral DNA was detected by Southern blot analysis with a [ 32 P]-labeled HPV18 DNA probe. DNA in lane 1 was digested with an enzyme that linearizes HPV18-GFP::SH, while the DNA in lane 2 was left untreated. The three topological forms of DNA (supercoiled, nicked and linear) are indicated on the right.

Article Snippet: To generate HPV18 genomes containing a GFP-Zeocin cassette, the GFP-Zeocin fusion gene was PCR amplified from pSELECT-CGFP (Invivogen; cat. no. psetz-zgfpsh) incorporating flanking BglII and BclI restriction sites.

Techniques: Transfection, Selection, Cell Culture, Immunofluorescence, Irradiation, Southern Blot, Labeling

Journal: Scientific Reports

Article Title: Novel recombinant papillomavirus genomes expressing selectable genes

doi: 10.1038/srep37782

Figure Lengend Snippet: ( a ) Primary HFKs were infected with HPV18-Neo containing quasivirions (WT and E1mt) at a VGE/cell of 100 and selected with 50 μg/ml G418 selection for either seven days (short) or continuously until staining. After 16 days feeders were removed and keratinocyte colonies were stained with methylene blue. ( b ) Quantitation of colonies formed in panel ( a ) error bars show SEM (n = 3). ( c ) Primary foreskin keratinocytes were infected with HPV18-Neo containing quasivirions. 48 hours post infection, cells were selected with 200 μg/ml G418 for an additional 96 hours after which selective pressure was removed. Keratinocyte colonies were stained with methylene blue. Infections were done in the presence of serum from rabbits vaccinated with the HPV vaccine Cervarix™ (or pre-immune control). Keratinocyte plates are a representative image. ( d ) Quantitation of colonies formed in panel ( c ) error bars show SEM (n = 3).

Article Snippet: To generate HPV18 genomes containing a GFP-Zeocin cassette, the GFP-Zeocin fusion gene was PCR amplified from pSELECT-CGFP (Invivogen; cat. no. psetz-zgfpsh) incorporating flanking BglII and BclI restriction sites.

Techniques: Infection, Selection, Staining, Quantitation Assay, Control