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

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.

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

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 436 ◽  
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.

scholarly journals The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay

2019 ◽  
Author(s):  
Kai Aoki ◽  
Takehito Sugasawa ◽  
Kouki Yanazawa ◽  
Koichi Watanabe ◽  
Tohru Takemasa ◽  
...  
Keyword(s):  
Whole Blood ◽  
Plasmid Vector ◽  
Sybr Green ◽  
Gene Doping ◽  
Human Erythropoietin ◽  
World Anti Doping Agency ◽  
Total Dna ◽  
Stool Dna ◽  
Tail Tip ◽  
Taqman Qpcr

BACKGROUND. With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. METHODS. Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 μL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. RESULTS. In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. CONCLUSIONS. These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

scholarly journals The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8595 ◽  
Author(s):  
Kai Aoki ◽  
Takehito Sugasawa ◽  
Kouki Yanazawa ◽  
Koichi Watanabe ◽  
Tohru Takemasa ◽  
...  
Keyword(s):  
Whole Blood ◽  
Plasmid Vector ◽  
Sybr Green ◽  
Gene Doping ◽  
Human Erythropoietin ◽  
World Anti Doping Agency ◽  
Total Dna ◽  
Stool Dna ◽  
Tail Tip ◽  
Taqman Qpcr

Background With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. Methods Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 µL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. Results In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. Conclusions These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

scholarly journals The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay

2019 ◽  
Author(s):  
Kai Aoki ◽  
Takehito Sugasawa ◽  
Kouki Yanazawa ◽  
Koichi Watanabe ◽  
Tohru Takemasa ◽  
...  
Keyword(s):  
Whole Blood ◽  
Plasmid Vector ◽  
Sybr Green ◽  
Gene Doping ◽  
Human Erythropoietin ◽  
World Anti Doping Agency ◽  
Total Dna ◽  
Stool Dna ◽  
Tail Tip ◽  
Taqman Qpcr

BACKGROUND. With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. METHODS. Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 μL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. RESULTS. In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. CONCLUSIONS. These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

Reduction of Prion Infectivity in Packed Red Cells by Separation of Whole Blood and Washing of the Red Cell Fraction.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2890-2890
Author(s):  
Rodrigo Morales ◽  
Kimberley A. Buytaert-Hoefen ◽  
Eric T. Hansen ◽  
Dennis Hlavinka ◽  
Raymond Goodrich ◽  
...  
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Whole Blood ◽  
Red Cells ◽  
Cell Fraction ◽  
Health Concern ◽  
Red Cell ◽  
New Variant

Abstract Although prion diseases are rare in humans, the established link between a new variant form of CJD (vCJD) and the consumption of cattle meat contaminated by BSE have raised concerns about a possible outbreak of a large epidemic in the human population. Over the past few years, BSE has become a significant health concern in several countries, and it now seems apparent that vCJD can also be iatrogenically transmitted from human to human by blood transfusion. Exacerbating this state of affairs is the lack of a reliable test to identify individuals incubating the disease during the long and silent period from the onset of infection to the appearance of clinical symptoms. The purpose of this research study was to evaluate the effectiveness of separation of whole blood and washing of the red cell fraction for the removal of infectious scrapie prion protein (PrPSc) from red blood cell (RBC) suspensions. Samples of human, whole blood were spiked with 5 × 106 LD50 263K PrPSc. Analysis of the treated sample supernatants by Western blot revealed that approximately >88% of the PrPSc was removed with the initial plasma expression and the equivalent of 6% was detected in a saline wash (300 mL; 0.9% saline). The final sample of RBCs revealed no detectable levels of PrPSc by Western blots. Further analysis of the treated RBCs using the PMCA assay indicated detectable amounts of PrPSc only after 2 consecutive amplification rounds. Semi-quantitative analysis of PMCA amplification enabled us to estimate that the treated RBCs contained less than 1 × 104 LD50 PrPSc. This corresponded to removal levels exceeding ≥99% of spiked material in whole blood. These in vitro estimations were confirmed by in vivo infectivity studies in a hamster model of disease transmission. Results from in vivo studies displayed significant differences in the incubation periods of the spiked blood inoculated hamsters (100.1 ± 1.7) versus washed RBCs (135.8 ± 6.7). Moreover, a substantial difference in the attack rate (6/15: 40% in washed RBC, versus 13/13: 100% in spiked blood) further indicated a substantial removal of infectious prions. Comparison of this data with results of animals inoculated with different dilutions of infectious material, indicated a >99.94% reduction of infectivity. Washed, packed human red cells produced by this procedure were able to be stored in standard additive solutions (AS-3) for 42 days while still meeting all in vitro blood bank standards for acceptable red cell quality. Conclusion This data suggests that separation of plasma coupled with a simple, low volume wash of red cells may represent an efficient method to remove prions from red blood cell fractions, thus reducing possible infectivity of these products.

IMPDH activity in whole blood and isolated blood cell fraction for monitoring of cellcept-mediated immunosuppression

1999 ◽  
Vol 31 (1-2) ◽  
pp. 1115-1116 ◽  
Author(s):  
M Storck ◽  
D Abendroth ◽  
W Albrecht ◽  
H.W Sollinger
Keyword(s):  
Blood Cell ◽  
Whole Blood ◽  
Cell Fraction ◽  
Impdh Activity

scholarly journals Plasma parasitemia as assessed by quantitative PCR in relation to clinical disease severity in African adults with falciparum malaria with and without HIV co-infection

Infection ◽  
2020 ◽  
Vol 48 (3) ◽  
pp. 367-373
Author(s):  
Aase Berg ◽  
Sam Patel ◽  
Marit G. Tellevik ◽  
Christel G. Haanshuus ◽  
Ingvild Dalen ◽  
...  
Keyword(s):  
Blood Cell ◽  
Severe Malaria ◽  
Falciparum Malaria ◽  
Disease Severity ◽  
Quantitative Pcr ◽  
Univariate Analysis ◽  
Cell Fraction ◽  
Malaria Parasitemia ◽  
Tertiary Teaching ◽  
The Impact

Abstract Purpose When considering malaria disease severity, estimation of parasitemia in erythrocytes is important, but sometimes misleading, since the infected erythrocytes may be sequestered in peripheral capillaries. In African children and Asian adults with falciparum malaria, parasitemia as assessed by quantitative PCR (qPCR) in plasma seems to be a valuable indicator of disease severity, but data on African adults as well as the impact of co-infection with HIV is lacking. Methods In 131 patients with falciparum malaria in a public tertiary teaching hospital in Mozambique, plasma malaria parasitemia as assessed by qPCR, compared to qualitative malaria PCR in blood cell fraction, was related to malaria disease severity and HIV co-infection. Results Of the 131 patients with falciparum malaria, based on positive qualitative PCR in the blood cell fraction, 93 patients (72%) had positive malaria qPCR in plasma. Patients with severe malaria as defined by the WHO criteria had higher malaria quantitative plasma parasitemia (median 143 genomes/µL) compared to those with uncomplicated malaria (median 55 genomes/µL, p = 0.037) in univariate analysis, but this difference was attenuated after adjusting for age, sex and HIV co-infection (p = 0.055). A quarter of the patients with severe malaria had negative qPCR in plasma. Conclusions This study of adult African in-patients with falciparum malaria with and without HIV co-infection, neither confirms nor rejects previous studies of malaria qPCR in plasma as an indicator of disease severity in patients with falciparum malaria. There is a need for further and larger studies to clarify if parasitemia as assessed malaria qPCR in plasma could be a surrogate marker of disease severity in falciparum malaria.

scholarly journals Telomere length varies substantially between blood cell types in a reptile

2020 ◽  
Vol 7 (6) ◽  
pp. 192136 ◽  
Author(s):  
Mats Olsson ◽  
Nicholas J. Geraghty ◽  
Erik Wapstra ◽  
Mark Wilson
Keyword(s):  
Blood Cell ◽  
Telomere Length ◽  
Whole Blood ◽  
Phylogenetic Analyses ◽  
Natural Populations ◽  
Cell Types ◽  
Tissue Type ◽  
Telomeric Dna ◽  
Blood Cell Type ◽  
Widespread Phenomenon

Telomeres are repeat sequences of non-coding DNA-protein molecules that cap or intersperse metazoan chromosomes. Interest in telomeres has increased exponentially in recent years, to now include their ongoing dynamics and evolution within natural populations where individuals vary in telomere attributes. Phylogenetic analyses show profound differences in telomere length across non-model taxa. However, telomeres may also differ in length within individuals and between tissues. The latter becomes a potential source of error when researchers use different tissues for extracting DNA for telomere analysis and scientific inference. A commonly used tissue type for assessing telomere length is blood, a tissue that itself varies in terms of nuclear content among taxa, in particular to what degree their thrombocytes and red blood cells (RBCs) contain nuclei or not. Specifically, when RBCs lack nuclei, leucocytes become the main source of telomeric DNA. RBCs and leucocytes differ in lifespan and how long they have been exposed to ‘senescence' and erosion effects. We report on a study in which cells in whole blood from individual Australian painted dragon lizards ( Ctenophorus pictus ) were identified using flow cytometry and their telomere length simultaneously measured. Lymphocyte telomeres were on average 270% longer than RBC telomeres, and in azurophils (a reptilian monocyte), telomeres were more than 388% longer than those in RBCs. If this variation in telomere length among different blood cell types is a widespread phenomenon, and DNA for comparative telomere analyses are sourced from whole blood, evolutionary inference of telomere traits among taxa may be seriously complicated by the blood cell type comprising the main source of DNA.

scholarly journals Assessment of Donkey (Equus asinus africanus) Whole Blood Stored in CPDA-1 and CPD/SAG-M Blood Bags

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 133
Author(s):  
Isabella Oliveira Barros ◽  
Rejane Santos Sousa ◽  
Marcondes Dias Tavares ◽  
Renato Otaviano Rêgo ◽  
Paulo Ricardo Firmino ◽  
...  
Keyword(s):  
Blood Cell ◽  
Cell Count ◽  
Whole Blood ◽  
Blood Collection ◽  
Blood Gas ◽  
Blood Cell Count ◽  
Animal Blood ◽  
Equus Asinus ◽  
Citrate Phosphate Dextrose ◽  
Citrate Phosphate

Hemotherapy using whole blood and its components is being increasingly used in veterinary therapy. Since it is important to store animal blood while maintaining acceptable hematological, blood gas, and biochemical characteristics, increasing our knowledge of available technologies for strategic blood storage is imperative. Thus, we aimed to assess the hematological, blood gas, and biochemical changes in donkey whole blood using blood bags with two different types of storage agents. Eight adult healthy male donkeys were used; 900 mL of blood was collected from each, with 450 mL stored in citrate-phosphate-dextrose and adenine bags (CPDA-1) and 450 mL stored in bags containing citrate-phosphate-dextrose, adenine, mannitol, and sodium chloride (CPD/SAG-M). Both bags were kept refrigerated between 1 and 6 °C for 42 days. Blood samples were removed from the bags eight times (T): T0 (immediately after blood collection), T1, T3, T7, T14, T21, T35, and T42 (1, 3, 7, 14, 21, 35 and 42 days after storage). Hematological, blood gas, biochemical, and microbiological parameters were assessed. The CPDA-1 bags had a higher packed cell volume when compared to CPD/ SAG-M. The red blood cell count reduced by around 19% in both the bags due to hemolysis, which was confirmed by an increase in plasma hemoglobin. The white blood cell count; pH; concentrations of glucose, sodium, bicarbonate, and 2,3 diphosphoglycerate were reduced in both bags. Meanwhile, pO2, pCO2, lactate dehydrogenase, and levels of potassium increased in the CPDA-1 and CPD/SAG-M bags. Blood bags were efficient for the storage of donkey blood for up to 42 days.

In vitro effects of temperature on red blood cell deformability and membrane stability in human and various vertebrate species

2021 ◽  
pp. 1-10
Author(s):  
Adam Attila Matrai ◽  
Gabor Varga ◽  
Bence Tanczos ◽  
Barbara Barath ◽  
Adam Varga ◽  
...  
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Whole Blood ◽  
Mechanical Stability ◽  
Cell Deformability ◽  
Blood Samples ◽  
Effects Of Temperature ◽  
The Way ◽  
Rbc Deformability

BACKGROUND: The effects of temperature on micro-rheological variables have not been completely revealed yet. OBJECTIVE: To investigate micro-rheological effects of heat treatment in human, rat, dog, and porcine blood samples. METHODS: Red blood cell (RBC) - buffer suspensions were prepared and immersed in a 37, 40, and 43°C heat-controlled water bath for 10 minutes. Deformability, as well as mechanical stability of RBCs were measured in ektacytometer. These tests were also examined in whole blood samples at various temperatures, gradually between 37 and 45°C in the ektacytometer. RESULTS: RBC deformability significantly worsened in the samples treated at 40 and 43°C degrees, more expressed in human, porcine, rat, and in smaller degree in canine samples. The way of heating (incubation vs. ektacytometer temperation) and the composition of the sample (RBC-PBS suspension or whole blood) resulted in the different magnitude of RBC deformability deterioration. Heating affected RBC membrane (mechanical) stability, showing controversial alterations. CONCLUSION: Significant changes occur in RBC deformability by increasing temperature, showing inter-species differences. The magnitude of alterations is depending on the way of heating and the composition of the sample. The results may contribute to better understanding the micro-rheological deterioration in hyperthermia or fever.

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