A Quantitative PCR Screening Method for Adeno‐Associated Viral Vector 2‐mediated Gene Doping

ABSTRACT We have cloned the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli. Here, we have subjected the HCMV BAC to random transposon (Tn) mutagenesis using a Tn1721-derived insertion sequence and have provided the conditions for excision of the BAC cassette. We report on a fast and efficient screening procedure for a Tn insertion library. Bacterial clones containing randomly mutated full-length HCMV genomes were transferred into 96-well microtiter plates. A PCR screening method based on two Tn primers and one primer specific for the desired genomic position of the Tn insertion was established. Within three consecutive rounds of PCR a Tn insertion of interest can be assigned to a specific bacterial clone. We applied this method to retrieve mutants of HCMV envelope glycoprotein genes. To determine the infectivities of the mutant HCMV genomes, the DNA of the identified BACs was transfected into permissive fibroblasts. In contrast to BACs with mutations in the genes coding for gB, gH, gL, and gM, which did not yield infectious virus, BACs with disruptions of open reading frameUL4 (gp48) or UL74 (gO) were viable, although gO-deficient viruses showed a severe growth deficit. Thus, gO (UL74), a component of the glycoprotein complex III, is dispensable for viral growth. We conclude that our approach of PCR screening for Tn insertions will greatly facilitate the functional analysis of herpesvirus genomes.

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The Third Replicon of Members of the Burkholderia cepacia Complex, Plasmid pC3, Plays a Role in Stress Tolerance

Applied and Environmental Microbiology ◽

10.1128/aem.03330-13 ◽

2013 ◽

Vol 80 (4) ◽

pp. 1340-1348 ◽

Cited By ~ 23

Author(s):

Kirsty Agnoli ◽

Carmen Frauenknecht ◽

Roman Freitag ◽

Stephan Schwager ◽

Christian Jenul ◽

...

Keyword(s):

Burkholderia Cepacia ◽

Screening Method ◽

Burkholderia Cepacia Complex ◽

Chromosome 3 ◽

Epidemic Strain ◽

Content Type ◽

Pcr Screening ◽

The Third ◽

Content Model ◽

Induced Stresses

ABSTRACTThe metabolically versatileBurkholderia cepaciacomplex (Bcc) occupies a variety of niches, including the plant rhizosphere and the cystic fibrosis lung (where it is often fatal to the patient). Bcc members have multipartite genomes, of which the third replicon, pC3 (previously chromosome 3), has been shown to be a nonessential megaplasmid which confers virulence and both antifungal and proteolytic activity on several strains. In this study, pC3 curing was extended to cover strains of 16 of the 17 members of the Bcc, and the phenotypes conferred by pC3 were determined.B. cenocepaciastrains H111, MCO-3, and HI2424 were previously cured of pC3; however, this had not proved possible in the epidemic strain K56-2. Here, we investigated the mechanism of this unexpected stability and found that efficient toxin-antitoxin systems are responsible for maintaining pC3 of strain K56-2. Identification of these systems allowed neutralization of the toxins and the subsequent deletion of K56-2pC3. The cured strain was found to exhibit reduced antifungal activity and was attenuated in both the zebrafish and theCaenorhabditis elegansmodel of infection. We used a PCR screening method to examine the prevalence of pC3 within 110 Bcc isolates and found that this replicon was absent in only four cases, suggesting evolutionary fixation. It is shown that plasmid pC3 increases the resistance ofB. cenocepaciaH111 to various stresses (oxidative, osmotic, high-temperature, and chlorhexidine-induced stresses), explaining the prevalence of this replicon within the Bcc.

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Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR

10.20944/preprints201904.0285.v1 ◽

2019 ◽

Author(s):

Takehito Sugasawa ◽

Kai Aoki ◽

Koichi Watanabe ◽

Koki Yanazawa ◽

Tohru Natsume ◽

...

Keyword(s):

Blood Cell ◽

Quantitative Pcr ◽

Whole Blood ◽

Digital Pcr ◽

Cell Fraction ◽

Detection Methods ◽

Gene Doping ◽

World Anti Doping Agency ◽

Injection Methods ◽

Tail Tip

With the rapid progress of genetic engineering and gene therapy, World Anti-Doping Agency has alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here we aimed to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. rAdV vectors containing mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could directly be detected from blood cell fraction-DNA, plasma-cell free DNA and stool-DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction-DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.

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Single-step cloning-screening method: a new tool for developing and studying high-titer viral vector producer cells

Gene Therapy ◽

10.1038/gt.2015.44 ◽

2015 ◽

Vol 22 (9) ◽

pp. 685-695 ◽

Cited By ~ 6

Author(s):

A F Rodrigues ◽

A S Formas-Oliveira ◽

M R Guerreiro ◽

H A Tomás ◽

P M Alves ◽

...

Keyword(s):

Viral Vector ◽

High Titer ◽

Screening Method ◽

Single Step

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Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR

Genes ◽

10.3390/genes10060436 ◽

2019 ◽

Vol 10 (6) ◽

pp. 436 ◽

Cited By ~ 3

Author(s):

Takehito Sugasawa ◽

Kai Aoki ◽

Koichi Watanabe ◽

Koki Yanazawa ◽

Tohru Natsume ◽

...

Keyword(s):

Blood Cell ◽

Quantitative Pcr ◽

Whole Blood ◽

Digital Pcr ◽

Cell Fraction ◽

Detection Methods ◽

Gene Doping ◽

World Anti Doping Agency ◽

Injection Methods ◽

Tail Tip

With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.

Download Full-text