A retroviral recombinant that highly expresses siRNA targeting the ATP binding region of bcrp/abcg2 gene
A high-efficiency expression and recombinant technology, applied in the field of genetic engineering, to achieve the effect of improving the drug-resistant phenotype
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Embodiment 1
[0015] Example 1 Design of RNA interference oligonucleotide fragments targeting the ATP-binding region of BCRP / ABCG2 gene.
[0016] Using Invitrogen's online software BLOCK-iT RNAi Designer, 3 sets of DNA oligonuclei complementary to the ABC structure region of BCRP / ABCG2 gene were designed for the nucleotide fragment cloning site of Invitrogen's pLenti6 / BLOCK-iT Lentiviral RNAi expression system vector The nucleotide chains, synthesized by Dalian Bao Bioengineering Co., Ltd., start from the 207th, 745th and 939th starting sites of the sixth exon of the BCRP / ABCG2 gene, respectively named BCRP1i, BCRP2i, BCRP3i .
[0017] The sequence of the sense strand of BCRP1i: 5'-CAGGATAAGCCACTCATAGA-3'; the sequence of the antisense strand is:
[0018] 5'-TCTATGAGTGGCTTATCCTG-3'.
[0019] The sequence of the sense strand of BCRP2i is: 5'-CAGATGCCTTCTTCGTTATG-3'; the sequence of the antisense strand is: 5'-CATAACGAAGAAGGCATCTG-3'.
[0020] The sequence of the sense strand of BCRP3i is:...
Embodiment 2
[0021] Example 2 Preparation of Retroviral Recombinant and Titration of Its Titer.
[0022] The retroviral RNAi expression system was recombined with oligonucleotide fragments, and the obtained recombinant retroviral expression RNAi vector was transfected into human embryonic kidney cells (293FT, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences), packaged into retroviral recombinants, and named V-BCRP1i, V-BCRP1i-C, V-BCRP2i, V-BCRP2i-C, V-BCRP3i, V-BCRP3i-C. Subsequently, the retroviral recombinant was infected with human choriocarcinoma cells (JAR, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences), and the titer was titrated, and the number of blue cell clones was obtained by dyeing with a violet dye for titer calculation. Titration result is as shown in table 1, each group retrovirus titer all reached 10 6 Grade, high infectivity.
[0023]
Embodiment 3
[0024] Example 3 RT-PCR detection of V-BCRPi interfering with BCRP / ABCG2 mRNA expression in JAR cells.
[0025] After JAR cells were treated with each retroviral recombinant V-BCRPi for 48 hours, total RNA was extracted using Invitrogen’s Total RNA Extraction Kit / Trizol Reagent for fluorescent quantitative RT-PCR detection. The experiment was repeated three times, and the results of one of them were as follows: figure 1 shown. figure 1 Among them, 2: pLenti6 / vector; 3: V-BCRP1i; 4: V-BCRP1i-c; 5: V-BCRP2i; 6: V-BCRP2i-c; 7: V-BCRP3i; 8: V-BCRP3i-c; The mRNA expression of V-BCRP3i was significantly different from that of the blank cell group ( P <0.01).
[0026] From figure 1 It can be seen that the mRNA expression of BCRP / ABCG2 in the cells of each retroviral recombinant V-BCRPi treatment group was significantly lower than that of the blank cell group; Compared with the respective V-BCRPi groups, the mRNA expression of BCRP / ABCG2 in the V-BCRPi-c group was slightly higher,...
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