RNA (ribonucleic acid) interference target points for inducing hepatocellular injury
An RNA interference and target technology, applied in DNA/RNA fragments, recombinant DNA technology, and cells modified by introducing foreign genetic material, etc., can solve the problems of blocked tyrosine catabolism, liver damage, and inability to generate fumaric acid.
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Embodiment 1
[0086] Example 1. Design and Construction of siRNA Expression Plasmids Targeting FAH
[0087] Selection and design of RNA interference targets targeting FAH: Sequence alignment of human and mouse FAH reference sequences, selection of regions with sequence differences (at least 1 base difference) between the two, step The sequence of the siRNA targeting FAH was designed by shifting; the siRNA sequence obtained from the primary selection was searched by BLAST in GenBank, and the sequence with 3 or more base differences from the non-targeting sequence was selected as the candidate sequence.
[0088] Construction of siRNA expression plasmid: In this example, pSUPER vector (Cat. No VEC-PBS-0001 / 0002 from oligoengine company) was used as an example to construct an siRNA expression plasmid. For the specific construction process, please refer to the company's pSUPER Vector Experiment Guide (www.oligoengine.com), and the brief construction process can be seen in the diagram figure 1 ....
Embodiment 2
[0090] Example 2. Co-transfection experiment screening to obtain RNA interference targets that can inhibit FAH
[0091] The deficiency of fumaryl acetoacetate hydrolase (FAH) will lead to the blockage of tyrosine catabolism, unable to generate fumarate, acetoacetate and succinate, causing the accumulation of tyrosine in the body, leading to liver damage (Grompe M. et al., Gene and Development, 1993, Vol. 7: pp. 2298-2307). Down-regulate the expression of FAH by RNA interference, in order to obtain the effect of liver injury.
[0092] pGL-FAH plasmid (construction method: by inserting the synthetic FAH gene sequence (GenBank No.NM_010176. 3) The reporter plasmid pGL3-FAH) was constructed as a plasmid capable of transcribing mRNA containing the luciferase-FAH sequence in mammalian cells (such as 293FT cells). If an siRNA that effectively targets FAH and cleaves the luciferase-FAH mRNA is present in the cell, translation of the luciferase-FAH mRNA can be prevented and a decreas...
Embodiment 3
[0100] Example 3. Construction of recombinant viral expression vector expressing siRNA
[0101] In this example, a recombinant lentiviral expression vector capable of expressing siFAH8 and siFAH10 was constructed.
[0102] Expression vector: In this example, the expression vector pDEST-MR of the lentiviral system we used (patent application number: 200510112917.1; publication number: CN1948475) contains an expression cassette that can be used to express siRNA controlled by the H1 promoter.
[0103] Construction of expression vectors pDEST-siFAH8 and pDEST-siFAH10:
[0104] Digest pSUPER-siFAH8 and pSUPER-siFAH10 (pSUPER vectors containing siFAH8 and siFAH10 respectively, see Example 1 for the construction method) with XhoI and XbaI to obtain nucleic acid fragments carrying siFAH8 and siFAH10 under the control of the H1 promoter; The obtained fragments were further ligated into the pDEST-MR vectors treated with the same restriction enzymes to construct expression vectors pDEST...
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