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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

Inactive Publication Date: 2011-12-21
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports on RNA interference that can efficiently inhibit the function of BCRP / ABCG2 at home and abroad

Method used

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  • A retroviral recombinant that highly expresses siRNA targeting the ATP binding region of bcrp/abcg2 gene
  • A retroviral recombinant that highly expresses siRNA targeting the ATP binding region of bcrp/abcg2 gene
  • A retroviral recombinant that highly expresses siRNA targeting the ATP binding region of bcrp/abcg2 gene

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to a retroviral recombinant capable of efficiently expressing siRNA targeting the ATP binding region of BCRP / ABCG2 gene, which belongs to the field of genetic engineering. Retroviral recombinants are recombined with retroviral RNAi expression system and oligonucleotide fragments, and are packaged by viral packaging cells. The invention is mainly applied to the drug resistance reversal of the breast cancer resistance protein.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a retroviral recombinant capable of efficiently expressing siRNA targeting the ATP binding region of BCRP / ABCG2 gene. Background technique [0002] The ultimate goal of studying tumor drug resistance mechanisms is to find targets for drug resistance formation, screen specific reversal agents, improve population disease prevention and clinical tumor chemotherapy effects, and reduce drug toxicity. For membrane protein transport substrates, researchers have a consensus: ATP is required to participate in the binding of target sequences and provide energy directly or indirectly through hydrolysis. Not only the formation of BCRP / ABCG2 transport drug function depends on the energy provided by ATPase hydrolysis of ATP, but also P- Classical membrane transporters such as gp also need the energy provided by ATPase to hydrolyze ATP, because they belong to the ATP-binding casse...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/113
Inventor 袁建辉刘建军徐新云柯跃斌程锦泉庄志雄
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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