Antigen-Specific Immune Response after Vaccination with TRP-2 and gp100 mRNA

gp100-TRP2

Immunization with mRNA coding for gp100 and TRP-2 in a lipid nanoparticle formulation induces an antigen-specific immune response.

mRNA coding for TRP-2 and gp100 were formulated in lipid nanoparticles and injected into C57BL/6 mice (n=5 for each group; 10 µg mRNA per mouse; subcutaneous injection; day 0). The vaccine was reapplied on days 30 and 33. Mice were bled on day 37 and splenocytes were isolated, stained with iTAg Tetramer/PE – H-2 Kb TRP2(SVYDFFVWL) and H-2Db gp100 Tetramer-EGSRNQDWL-PE respectively, and analyzed by flow cytometry. The 3 immunizations resulted in 15% of antigen specific CD 8 T cells for the gp100 mRNA and 22% for the TRP-2 mRNA respectively.

Submitted by TriLink client.

Robust in vivo expression after injection of Nanotaxi®/mRNA

luciferase-expression

Graphe-EPO

(Top) Luciferase expression in muscle after injection of Nanotaxi®/mRNA. Luciferase was observed by bioimaging at day 1. (Bottom) Modulation of the hematocrit level in mice intramuscularly injected with Nanotaxi®/mRNA over a period of 6 months. At day 0 and 42, mice received treatment#1 and #2 consisting of 2 successive injections with one week interval of mRNA encoding murine EPO (mEPO) either naked (open triangles) or formulated with Nanotaxi®1 (solid squares) or Nanotaxi®2 (solid circles). At day 100 and 134, mice received respectively treatment#3 consisting of mEPO mRNA either naked or formulated with Nanotaxi®1 or Nanotaxi®2, and treatment#4 consisting of mEPO mRNA formulated with Nanotaxi®1. As control, mice were also uninjected (open squares). Dotted lines represent the fluctuation of the physiological hematocrit level. Cat #: L-6107, L-6118.

Data courtesy of In-Cell-Art.

EGFP mRNA exhibits high cellular expression in multiple cell lines using mRNA-In™

hela human huvec mess sh-sy

Cells were split into 24-well plates to give 60-70% confluence the day of transfection. EGFP mRNA was complexed with various amounts of mRNA-In™ in a total of 50µl of OptiMEM. mRNA /mRNA-In™ complexes were added to the cells, mixed, and incubated at 37ºC. Cells were observed at 24 hours post-transfection. Cat #: L-6101

Data courtesy of Donna R. Trollinger, Ph.D., MTI-GlobalStem

Ovalbumin mRNA Vaccination Stimulated CD4 and CD8 T Cell Proliferation and Resulted in a High Number of Antigen Specific CD8 T Cells

four

OT-II CD 4 T cells and OT-I CD 8 T cells specifically recognize epitopes of ovalbumin. OT-I and OT-II T cells were labeled with intracellular carboxyfluorescein succimidyl ester (CFSE) dye. The dye serves as an indicator of T cells activation/division. With every cell divisions, the dye gets distributed between both daughter cells and hence diluted to half.

One million OT-I and one million OT-II T cells were injected into wild type C57BL/6 mice. Mice were immunized the next day by subcutaneous injection with Ovalbumin (10 mg in PBS), OVA mRNA (30 μg, formulated in lipid nano particle), or OVA mRNA modified with 5meC, PseudoU (30 μg, formulated in lipid nanoparticle). The negative control was naïve mice that were not immunized. Three mice per treatment group were used. The draining lymph nodes were harvested four days after immunization and cells were stained and analyzed by flow cytometry. Cat #: L-6128, L-6328.

Data submitted by TriLink client.

Cas9 and Cas9 nickase mRNA efficiently cleave genomic DNA

gell

80,000 HEK293T cells/ well were seeded in a 24 well plate the day before transfection with TransIT® mRNA transfection reagent (Mirus Bio). % indel formation is indicated.  (Nick) Cells were transfected with 500 ng of Cas9 nickase mRNA, 250 ng of guide strand plasmid A plus 250 ng of guide strand plasmid B. (Nuc) 500 ng of Cas9 mRNA was transfected along with 500 ng guide strand plasmid. After 72 hours, genomic DNA was harvested from each well and amplified with primers flanking the target site. Cleavage and misrepair was estimated using the T7E1 mutation detection assay and separation on an agarose gel. Cat #: L-6125, L-6116.

Image courtesy of TJ Cradick / Gang Bao, Georgia Tech.

Transfection of Cas9 mRNA and guide strands leads to high genome editing efficiency

293 FT cells

Human embryonic kidney (HEK) cell line 293FT (Life Technologies) was maintained in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2mM GlutaMAX™ (Life Technologies), 100U/mL penicillin, and 100μg/mL streptomycin at 37°C with 5% CO2 incubation.

293FT cells were seeded onto 24-well plates 24 hours prior to transfection at the density of 120,000 cells per well. Cells were transfected using Lipofectamine® 2000 (Life Technologies) at 80-90% confluency following the manufacturer’s recommended protocol. For each well of a 24-well plate, either 400ng Cas9 DNA + 100ng sgRNA, or 1000ng of Cas9 mRNA + 100ng sgRNA was used.

Cells were harvested 72-hours post transfection for DNA extraction. Indel detection was performed using the SURVEYOR Mutation Detection kit from Transgenomic. The bottom two bands designated by arrows provide evidence for CRISPR mediated genome cleavage followed by imperfect repair by non-homologous end joining. Cat #: L-6112.

Image courtesy of Feng Zhang, Broad Institute of MIT and Harvard.