Heterogeneous CIC cell model of targeted adhesion molecule and preparation method thereof
A cell model and target cell technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, and cells modified by the introduction of foreign genetic material, etc., can solve the problem of lack of targeted specific gene models, and achieve the promotion of immune killing. , the effect of inhibiting tumor growth
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[0056] Example 1. Establishment of Inhibitory Heterogeneous CIC Cell Model
[0057] The target cell in this example is PLC / PRF / 5 liver cancer cell monoclonal line F6ft knockdown CD44 gene cell line shCD44-F6ft (high CIC formation rate), Western blot can detect N-cadherin and β-catenin genes Expression of the encoded protein in this cell line ( figure 1 B, C), please refer to Western blot in step one 2 of this example for details.
[0058] 1. Knock down the expression of N-cadherin and β-catenin genes
[0059] 1. Knock down N-cadherin and β-catenin genes by siRNA
[0060] 1) Design of siRNA: Two siRNA sequences targeting N-cadherin and β-catenin genes were designed and synthesized by Suzhou Gemma Company.
[0061] 2) Transfection of siRNA: using a transfection kit RNAiMAX Reagent (Invitrogen, #13778) was used for siRNA transfection, and the reaction system and conditions were operated according to the instructions of the kit. The details are as follows: Prepare 1×10 5 He...
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[0096] Example 2. Establishment of induced heterogeneous CIC cell model
[0097] The target cell in this example is the PLC / PRF / 5 liver cancer cell monoclonal line F6ft. Western blot can detect that the expression of N-cadherin and β-catenin gene-encoded proteins is low ( figure 2 B, C), please refer to Western blot in step one 2 of this example for details.
[0098] 1. Establishment of F6ft-Ncadherin and F6ft-βcatenin overexpressing cell lines
[0099] 1. Construction of a plasmid overexpressing N-cadherin
[0100] 1) PCR amplification of N-cadherin fragment: PCR amplification of the N-cadherin gene coding sequence fragment CDS with Xho I and EcoR I restriction sites using cDNA as a template, and a gel DNA fragment recovery kit (Tiangen, #DP208-02) for purification.
[0101] 2) Enzyme digestion: The pQCXIP-mCherry-N1 backbone plasmid and the PCR-amplified N-cadherin fragment were mixed with restriction enzymes Xho I, EcoR I (New England Biolabs) and reaction buffer, and p...
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