Unreliable Real-Time PCR Analysis of Human Endogenous Retrovirus-W (HERV-W) RNA Expression and DNA Copy Number in Multiple Sclerosis

We used quantitative real-time PCR analysis to measure the copy number of the BARE-1 retrotransposon in 5 cultivars of barley (Hordeum vulgare), as well as in samples from its wild relative, Hordeum spontaneum. Two sets of PCR primers were used to amplify regions within the long terminal repeat (LTR) and the reverse transcriptase (RT) gene of BARE-1 (GenBank accession Z17327). The LTR primers detected an average of 2.148 × 105 ± 0.012 × 105 copies per haploid genome among barley samples, whereas the RT primers detected an average of 1.588 × 104 ± 0.085 × 104 copies. The average ratio of LTR:RT was estimated to be 13.5:1. This finding indicates that more than 7% of the barley genome is occupied by BARE-1 elements in the form of solo LTRs and another 2.6% of the genome is occupied by the full-length element. Taken together, BARE-1 sequences represent approximately 9.6% of the barley genome among the barley plants used in this study. For the above estimation, a genome size of 5.44 × 103 Mb for H. vulgare and 5.39 × 103 Mb for H. spontaneum were assumed. Our study on quantification results of the BARE-1 for a small group of barley cultivars showed that there are significant differences among cultivars in terms of BARE-1 copy number, providing further evidence that BARE-1 is active and has a major role in shaping the barley genome as a result of breeding and selection. Quantification results also showed that most of the elements (> 90%) are present as truncated copies (solo LTRs). These results show that there is a high level of recombination leading to the formation of truncated elements and a subsequent DNA loss from the genome. Taken together, our study provides a glimpse into a dynamic micro-evolutionary process that is the by-product of genome reshuffling and directional selection in barley breeding programsKey words: BARE-1, genome evolution, quantification, real-time PCR, retrotransposons.

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Retrovirus Insertion Site Analysis Following In Vivo Drug Selection in a Phase I Clinical Trial Utilizing G156A MGMT for Enhanced Chemotherapy of Advanced Malignancies.

Blood ◽

10.1182/blood.v106.11.3048.3048 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3048-3048

Author(s):

Colin L. Sweeney ◽

Karen Lingas ◽

Jane S. Reese ◽

Susan Flick ◽

Stanton L. Gerson

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Insertion Site ◽

Pcr Analysis ◽

Site Analysis ◽

Cd34 Cells ◽

Vector Copy Number ◽

Post Infusion

Abstract The G156A mutant of the DNA repair gene O6-methylguanine DNA-methyltransferase (MGMT) confers hematopoietic resistance to O6-benzylguanine (BG) combined with DNA-alkylating agents BCNU or temozolomide, and allows for selective in vivo expansion with drug administration of murine hematopoietic progenitors transduced with G156A MGMT retrovirus. Here we report our latest findings on retroviral vector copy number and insertion site analysis following drug treatment from a Phase I clinical trial utilizing MGMT-mediated chemoprotection for enhanced treatment of advanced solid tumors. Seven patients have entered the trial and 6 have completed the cell infusion process. For all patients, autologous CD34+ cells were transduced ex vivo with an MFG retroviral vector containing the G156A MGMT gene (packaged with PG13 by the National Gene Vector Laboratory, Ken Cornetta, Director) in the presence of the fibronectin fragment CH-296 and the cytokines SCF, Tpo, and Flt-3 ligand for 72 hours with three additions of retroviral supernatant. At 72 hours following patient treatment with BG and BCNU, cells were re-infused. Prior to infusion, the average vector copy number by quantitative real-time PCR analysis for six patients was 0.34 copies per genome, with an average of 24% of CFUs transduced by standard PCR for G156A MGMT, and an average of 9% of CD34+ cells expressing the MGMT transgene by flow cytometry. In one patient with metastatic melanoma we have further analysis of insertions. For this patient, the pre-infusion vector copy number of the bulk CD34+ population was 0.54 copies per genome by real-time PCR, with 27% of CFUs transduced and 8% of CD34+ cells expressing the MGMT transgene prior to infusion. Linear amplification-mediated (LAM)-PCR analysis of retroviral insertion sites in pre-infusion CFUs from this patient confirmed a polyclonal population, with an average of 1.6 retroviral insertions per positive CFU. In this patient, BG (120 mg/m2) and BCNU (33 mg/m2) were administered at 6 weeks post-infusion, and temozolomide (300 mg/day for 5 days) was administered at 13 weeks. Peripheral blood (PB) and bone marrow (BM) granulocyte and mononuclear cells (MNCs) were collected at weeks 5, 11, 15, and 16 for DNA and CFU analysis. Vector copy number at all post-infusion time points was below the limit of detection of SYBR Green probe-based real-time PCR (<100 copies of G156A MGMT per 5000 genomes). LAM-PCR detected the vector in post-treatment samples based on an internal vector control band present in BM MNCs at week 11 and in BM granulocytes at week 16, although specific insertion sites were not detected. Standard PCR revealed 1 out of 100 CFUs from week 11 BM MNCs contained the vector, with 2 out of 30 CFUs from week 15 PB MNCs. LAM-PCR in a subset of week 11 CFUs confirmed a single insertion site present in the same PCR-positive CFU. Sequence analysis of clonal vector insertions pre- and post-infusion is ongoing, and thus far a number of sites have been characterized, adding to the emerging database of clinical retroviral insertions. These are the first data to show emergence of transduced mutant MGMT cells after nonmyeloablative conditioning in humans and suggest that despite a low frequency of vector-marked hematopoietic cells, clinical in vivo drug selection can be observed.

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Increased copy number of syncytin-1 in the trophectoderm is associated with implantation of the blastocyst

PeerJ ◽

10.7717/peerj.10368 ◽

2020 ◽

Vol 8 ◽

pp. e10368

Author(s):

Luyan Guo ◽

Fang Gu ◽

Yan Xu ◽

Canquan Zhou

Keyword(s):

Stem Cells ◽

Real Time ◽

Copy Number ◽

Embryo Implantation ◽

Endogenous Retrovirus ◽

Envelope Proteins ◽

Human Endogenous Retrovirus ◽

Relative Copy Number ◽

Relative Expression ◽

Real Time Qpcr

Background A key step in embryo implantation is the adhesion to and invasion of the endometrium by the blastocyst trophectoderm. The envelope proteins of HERV-W and -FRD (human endogenous retrovirus-W and -FRD), syncytin-1 and syncytin-2, are mainly distributed in the placenta, and play important roles in the development of the placenta. The placenta originates from the trophectoderm of the blastocyst. It is unclear whether the envelope proteins of HERV-W and -FRD have an effect on the development of the trophectoderm and whether they have any association with the implantation of the blastocyst. Methods The whole-genome amplification products of the human blastocyst trophectoderm were used to measure the copy number of syncytin-1 and syncytin-2 using real time qPCR. In addition, clinical data associated with the outcome of pregnancies was collected, and included age, body mass index (BMI), basic follicle stimulating hormone(bFSH), rate of primary infertility and oligo-astheno-teratospermia, the thickness of the endometrium on the day of endometrial transformation, the levels of estrogen and progestin on the transfer day, the days and the morphological scores of the blastocysts. The expression of mRNA and the copy numbers of syncytin-1 and syncytin-2 in H1 stem cells, and in differentiated H1 cells, induced by BMP4, were measured using real time qPCR. Results The relative copy number of syncytin-1 in the pregnant group (median: 424%, quartile: 232%–463%, p < 0.05) was significantly higher than in the non-pregnant group (median: 100%, quartile: 81%–163%). There was a correlation (rs = 0.681, p < 0.001) between the copy number of syncytin-1 and blastocyst implantation after embryo transfer. As the stem cells differentiated, the expression of NANOG mRNA decreased, and the expression of caudal type homeobox 2(CDX2) and β-human chorionic gonadotropin (β-hCG) mRNAs increased. Compared to the undifferentiated cells, the relative expression of the syncytin-1 mRNA was 1.63 (quartile: 0.59–6.37, p > 0.05), 3.36 (quartile: 0.85–14.80, p > 0.05), 10.85 (quartile: 3.39–24.46, p < 0.05) and 67.81 (quartile: 54.07–85.48, p < 0.05) on day 1, 3, 5 and 7, respectively, after the differentiation. The relative expression of syncytin-2 was 5.34 (quartile: 4.50–10.30), 7.90 (quartile: 2.46–14.01), 57.44 (quartile: 38.35–103.87) and 344.76 (quartile: 267.72–440.10) on day 1, 3, 5 and 7, respectively, after the differentiation (p < 0.05). The copy number of syncytin-1 increased significantly during differentiation. Conclusion Preceding the transfer of frozen embryos, the increased copy number of syncytin-1 in the blastocyst trophectoderm was associated with good outcomes of pregnancies.

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