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Use of single-stranded DNA in gene transfection

A gene transfection, single-stranded technology, applied in DNA preparation, recombinant DNA technology, microbial assay/inspection, etc., can solve the problems of easy degradation, difficult to obtain RNA, instability and so on

Inactive Publication Date: 2021-01-08
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

plDNA generally causes unnecessary gene or protein expression due to the introduction of carrier sequences. RNA is difficult to obtain, unstable, and easy to degrade. ASO also requires specific modifications to be stable. Currently, the nucleic acids commonly used for gene delivery have certain insufficient

Method used

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  • Use of single-stranded DNA in gene transfection
  • Use of single-stranded DNA in gene transfection
  • Use of single-stranded DNA in gene transfection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The EGFP cssDNA and EGFP-mCherry cssDNA produced based on the pScaf vector of this example are used for transfection of 293T cells, Hela cells, and A549 cells:

[0045] Step 1. Construction and extraction of cssDNA

[0046] (1) Insert the EGFP gene and EGFP-mCherry gene into the pScaf vector to construct the pScaf-EGFP vector and pScaf-EGFP- mCherry vector (such as image 3 shown);

[0047] (2) Co-transform 10ng pScaf-EGFP vector, 10ng pScaf-EGFP-mCherry vector and 15ng M13-based pSB4423 plasmid into XL1-Blue competent cells, plate, and culture at 30°C for 16h;

[0048] (3), pick a single clone in 1mL 2×YT (5mM MgCl 2 ) culture medium at 30°C, 220rpm for 1h;

[0049] (4), the above medium in 5mL 2 × YT (5mM MgCl 2 , 100 μg / mL ampicillin sodium, 20 μg / mL chloramphenicol) for 4 hours, 30 ° C, 220 rpm;

[0050](5) Cultivate the above bacterial liquid in 200mL 2×YT medium at 30°C and 220rpm, wherein pScaf-EGFP is cultivated for 8h, and pScaf-EGFP-mCherry is cultivated...

Embodiment 2

[0073] The linear single-stranded DNA produced based on asymmetric PCR in this example: EGFP lssDNA is used for transfection of 293T cells and MGC803 cells:

[0074] Step 1. Construction and extraction of lssDNA

[0075] (a), prepare the PCR reaction system according to the following formula:

[0076] Reverse product amplification: pEGFP-N1 plasmid: 2 μL (10ng / μL), upstream primer: 2 μL (0.2 μM), downstream primer: 2 μL (10 μM), PrimeSTAR Max Premix (25 μL, R045A, Takara); ddH 2 O: 19 μL.

[0077] Forward product amplification: pEGFP-N1 plasmid: 2 μL (10ng / μL), upstream primer: 2 μL (10 μM), downstream primer: 2 μL (0.2 μM), PrimeSTAR Max Premix (25 μL, R045A, Takara); ddH 2 O: 19 μL.

[0078] Wherein, the upstream primer sequence in forward product amplification and reverse product amplification is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4.

[0079] (b), amplify according to the following reaction:

[0080] React at 95°C for 10 min,...

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Abstract

The present invention provides a use of single-stranded DNA in gene transfection and particularly relates to a use of cyclic single-stranded DNA generated on the basis of a traditional phage vector and linear single-stranded DNA obtained by an asymmetric PCR method in gene transfection. Different from traditional transfection of cyclic double-stranded plasmids, transfection efficiency of the cyclic single-stranded DNA is in direct proportion to concentration of a transfection reagent, the cyclic single-stranded DNA with the same concentration is more easily delivered into cells difficult to transfect by the transfection reagent, the cyclic single-stranded DNA has higher expression quantity, causes low cytotoxicity and thus can obviously reduce the cytotoxicity caused by a polymer, effectively improves transfection efficiency, also does not obviously cause the cytotoxicity. Besides, the linear single-stranded DNA also proves the feasibility of gene transfection, so that expressions of related genes can be seen when the single-stranded DNA is used for gene transfection, thereby indicating that the single-stranded DNA can be used as a vector for the gene transfection.

Description

technical field [0001] The invention belongs to the technical field of gene delivery, and in particular relates to the application of single-stranded DNA in gene transfection. Background technique [0002] Traditional gene delivery refers to the introduction of plasmid DNA (plDNA), small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), antisense oligonucleotide (ASO), etc. A cellular or bodily process. plDNA generally causes unnecessary gene or protein expression due to the introduction of carrier sequences. RNA is difficult to obtain, unstable, and easy to degrade. ASO also requires specific modifications to be stable. Currently, the nucleic acids commonly used for gene delivery have certain insufficient. However, gene delivery for single strands has not been reported yet. Single strands have the advantages of being easy to be compressed, easy to obtain, and will not introduce too many nonsense sequences. Contents of the invention [0003] In view of th...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N15/64C12N15/10
CPCC12N15/85C12N15/66C12N15/64C12N15/10C12N2800/107C12Q2531/107C12Q2565/125C12N15/87Y02A50/30
Inventor 宋杰程进唐林林
Owner SHANGHAI JIAO TONG UNIV
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