Liver:For liver targeting, we need to first find a protein specifically recognize hepatic cells. Since Heptitis B virus can infect hepatic cells distinctively, and from recent study[1], we knew that HBV recognizes the hepatic cells via the interaction between the pre-S1 of the HBV envelop protein and NTCP receptor of the hepatic cells. We tried to engineer the pre-S1 from HBV envelope protein to the lamp 2b.
Therefore we cloned the pre-S1 into lamp 2b, and we choose pcDNA 3.1(+) as our backbone.
Results for liver targeting:To produce the exosomes that have pre-S1 on their surface for liver targeting, we first transfected the exosome-producing cells, HEK 293T cells, with the plasmid encoding the fusion protein of lamp 2b and pre-S1 peptide.
siRNA screening (Failed)14th: We extracted plasmids and cultured the 293t cells.15th: We transfected plasmids into 293t cells.16th: We collected cells and preserved it in Trizol.17th: We extracted RNA, then did RT-PCR with the RNA.18th: We did qPCR with the cDNA we got on 17th April.21th: We reexamined the concentration of RNA, and redid qPCR.
siRNA screening (Failed)27th: We cultured the 293t cells in a 12-well plate.28th: We transfected 293t cells.29th: We collected cells and extracted RNA from it.30th: We did RT-qPCR with the RNA we extracted on 29th April.1st: We did qPCR with the cDNA we got on 30th April.
siRNA screening (Failed), examination whether siRNA are capsulated into exosomes (Success)8th: We extracted plasmids of 3 kinds of siRNA.9th: We cultured 293t cells in eight D10 dishes and a 12-well plate.10th: We extracted 2 kinds of over-expression plasmids and examined the concentration of it. Then we transfect these plasmids into 293t cells respectively.11th: We collected cells and exosomes from 8 D10 dish, and cells from 12-well plate.12th: We extracted RNA from cells and exosomes.13th: We did RT-PCR and qPCR.
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