DNA methylation research grant winners

Diagenode offers grant programs giving researchers an opportunity to share their unique research with us and apply for a high-value grant. The researchers below were awarded product grants for DNA methylation studies in the value of $2,000/2,000 €.

Persistency of Transgene Expression Mediated by Lentiviral Gene Delivery in Pluripotent Cell Lines

My general objectives are to (1) determine the duration of reporter gene expression from pluripotent cell lines transduced by lentivirus and (2) to assess the epigenetic effects on the provirus. More specifically, I plan to (1) produce LV carrying Green Fluorescent Protein (GFP) reporter gene, (2) obtain and maintain the required cell lines including establishing primary mouse fibroblast as a control, 3) measure the duration of GFP expression from pluripotent and control cell lines transduced with the lentivirus, (4) exclude the loss of the integrated provirus as the factor for GFP silencing in transduced non-pluripotent cell lines, and (5) study the effects of epigenetics on the GFP gene and the regulatory sequence of the provirus.

Transgene integration by lentiviral (LV) vector in the host cell’s genome would theoretically generate a prolonged or permanent transgene expression. However, several citations have reported a decline in transgene expression in early progenitor cells and stem cells transduced by LV. We hypothesized that prolonged transgene expression can be achieved if the transgene is introduced into the cells before epigenetic markers are established in the genome, i.e during the pluripotency period. Therefore, the proposed study seeks to determine if this phenomenon would occur in pluripotent cell lines, focusing on mouse induced pluripotent stem (iPS) cells as the target cell, in a gene therapy context.

Two epigenetic analyses that will be performed on the promoter and transgene of the provirus are DNA methylation and chromatin modification. For DNA methylation profiling, the genomic DNA of the cell will be treated with bisulfite prior to PCR and sequencing of the proviral DNA. The cells may also be analyzed for 5-hmC using 5-hmC monoclonal antibody (C15200200-200) or hMeDIP Kit (C02010031) to assess the level of hydroxymethylation. We may also consider using Diagenode’s MethylCap kit (C02020010) to fractionate the methylated DNA by CpG density.

For the chromatin modification analysis, the cells will be treated with trichostatin A (TSA) before chromatin immunoprecipitation (ChIP) analysis by chromatin IP - bisulfite - sequencing (ChIP-Bis-Seq) and as well Combining chromatin IP and DNA methylation profiles in one assay using the Premium Bisulfite kit, Diagenode. We may also perform pull-down methylated DNA analysis by using specific antibodies such as (1) H3K4 monoclonal antibody (C1541065) (2) H3K4 polyclonal antibody (C15410037) (3) H3K9 polyclonal antibody (C15410004). Beads only will be used as a control.

Mr. Alhaji’s products of interest:

• 5hmC monoclonal antibody (C15200200)
• hMeDIP Kit (C02010031)
• MethylCap kit (C02020010)
• Premium Bisulfite Kit (C02030030)
• H3K9me1 monoclonal antibody (C15410065)
• H3K4me1 polyclonal antibody (C15410037)
• H3K9ac polyclonal antibody (C15410004)

Epigenome-wide methylation pattern discovery for irradiated skin in radiotherapy

Radiotherapy is utilized in neoadjuvant, definitive, and palliative treatment of a wide variety of cancers. From a reconstructive perspective, however, irradiated fields pose significant challenges as the tissue is often stiff, brittle, and heals poorly. Little is known about the mechanism by which irradiation produces these changes. The objective of our research is to reveal the epigenetic changes that occur in irradiated skin in order to identify potential targets for therapy. We aim to translate our findings into interventional studies in both cell culture and a mouse model to assess their efficacy in vitro and in vivo.

We have access to a bank of paired samples of irradiated and non-irradiated tissues from patients who have undergone reconstructive procedures after cancer treatment. We have used the Illumina Infinium Human Methylation450 BeadChip array to assess epigenome-wide methylation status of eight paired samples, and have identified a signature methylation pattern for irradiated skin. We would like to utilize the Diagenode Premium WGBS Kit for bisulfite sequencing of additional paired samples for two purposes. First, we would like to confirm the findings of our BeadChip array utilizing a more robust method of assessing epigenome-wide methylation status. Second, we would like to assess methylation status at loci that are not evaluated by the BeadChip array. Using this information, we plan to identify loci with the greatest change in methylation status between irradiated and non-irradiated samples. By comparing these loci to literature, we intend to identify genes that are known to have an effect on wound healing. We then plan to design interventional studies to assess the effects of modulating the expression of these genes and/or supplementing gene products in cell culture and a mouse model. We would like to utilize the Diagenode Bisulfite Kit to confirm methylation status at target genes in these additional studies.

Dr. Johnson’s products of interest:

• Premium WGBS Kit (C02030034)
• Premium Bisulfite Kit (C02030030)

Next-generation sequencing based methylome study of primary breast tumours

Breast cancer is one of the leading causes of cancer death in women, and is unanimously considered a heterogeneous disease displaying distinct therapeutic responses and outcomes. While recent advances have led to the refinement of the molecular classification of the disease, the epigenetic landscape has received less attention.

We are delighted to win the DNA methylation research grant award and intend to use it to conduct a next-generation sequencing based methylome study of primary breast tumours. DNA methylation markers will also be investigated in Patient Derived Tumour Xenografts (PDTXs) and in circulating tumour DNA (ctDNA) to identify potential prognostic and predictive methylation biomarkers in breast cancer.

Mr. Batra’s products of interest:

• Premium WGBS Kit (C02030034)
• Premium RRBS Kit (C02030032)