Lentiviral vector expression system for polygene transformation

A lentiviral vector and expression system technology, applied in the direction of virus/bacteriophage, reverse transcription RNA virus, introduction of foreign genetic material using vectors, etc., can solve the problems of harsh cells, achieve the effect of improving the expression rate and avoiding environmental pollution

Inactive Publication Date: 2021-10-26
SHANDONG VIGENE BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, selection with multiple antibiotics at the same time is often harsh on the cells

Method used

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  • Lentiviral vector expression system for polygene transformation
  • Lentiviral vector expression system for polygene transformation
  • Lentiviral vector expression system for polygene transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Construction of the first and second lentiviral vectors

[0081] 1. Construction of vectors pLent-EF1a-MCS-CMV-N-Puro-TS1 and pLent-EF1a-MCS-CMV-TS2-C-Puro

[0082] 1. Design primers. See Table 3 for specific primers:

[0083] Table 3: PCR Primers

[0084]

[0085] 2. PCR, obtain N-Puro-TS1 fragment and TS2-C-Puro fragment by PCR technique.

[0086] Table 4: PCR system: (unit: μL)

[0087]

[0088] Reaction procedure:

[0089] Table 5: PCR program

[0090]

[0091] After the PCR reaction, take 2 µl of the PCR product and use 1.5% agarose gel electrophoresis to separate and detect it. If the size of the target band is correct, use the PCR purification recovery kit to recover the target fragment.

[0092] 3. Digestion

[0093] (1) Digest the N-Puro-TS1 and TS2-C-Puro obtained by PCR, and screen the enzyme digestion system as shown in Table 6:

[0094] Table 6: Enzyme digestion system for screening genes

[0095]

[0096] (2) After adding th...

Embodiment 2

[0157] Example 2: Vector transfected cells, target gene transcription

[0158] The constructed pLent-EF1a-Oct4-P2A-Sox2-CMV-N-Puro-TS1 (P3) vector and pLent-EF1a-Klf4-P2A-Myc-CMV-TS2-C-Puro (P4) vector were co-transfected into HEK293 Cells, puro screening for 72 hours of viability verification and detection of mRNA levels of four genes

[0159] 1. The constructed pLent-EF1a-Oct4-P2A-Sox2-CMV-N-Puro-TS1 vector and pLent-EF1a-Klf4-P2A-Myc-CMV-TS2-C-Puro vector were co-transfected into HEK293 cells, puro screening 72 hours of viability verification

[0160] 1.1 Normal-growing HEK293 cells were spread in 6-well plates;

[0161] 1.2 Carry out transfection experiments according to Table 17:

[0162] 1.2.1 Cell culture: take 6-well plate cell culture dish, add 2mL containing 1~2×10 5 cell culture medium at 37°C CO 2 The culture density is 40%~60%, if the density is too high, it is not easy to select cells after transfection.

[0163] 1.2.2 Preparation of transfection solution A...

Embodiment 3

[0186] Example 3: Infection of 293A cells with packaging lentivirus and selection of stably transfected cells

[0187] 3.1 Packaging lentiviral products:

[0188] 3.1.1 Spread HEK293T cells in a 10cm dish, and the next day the cell confluency reaches 85%-90%;

[0189] 3.1.2 Configure the transfection reagent according to the ratio, mix it and let it stand at room temperature for 3 minutes, then add PEI and vortex to mix, let it stand at room temperature for 30 minutes, add it to a 10cm cell culture dish, and replace it with fresh medium 6 hours after transfection;

[0190] 3.1.3 Observe the transfection efficiency and take pictures;

[0191] 3.1.4 Collect the supernatant medium after 72 hours of transfection, pass through a 0.45um filter membrane; put the filtered supernatant medium into an ultrafiltration tube for ultracentrifugation, 25000rpm, 4°C, 1.5hs;

[0192] 3.1.5 Collect viruses.

[0193] 3.2 Detection of virus titer:

[0194] 3.2.1 6 hours before infection in 24-...

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Abstract

The invention provides a lentiviral vector expression system for realizing polygene transformation by splitting a single selection marker, and comprises the lentiviral vector expression system, a construction method thereof and application in the polygene transformation. By use of the lentiviral vector expression system provided by the invention, transformation of a plurality of target genes can be realized by using the single selection marker, and the packaging rate of the expression system and the expression rate of the target genes can be improved in various cells.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a lentiviral vector expression system for realizing multigene transformation by splitting a single screening marker, including the lentiviral vector expression system, its construction method and application in multigene transformation. Background technique [0002] Lentiviral vector is a virus vector system modified on the basis of HIV-1 virus, which can efficiently introduce target genes into animal and human primary cells or cell lines. Lentiviral vector-mediated gene expression is sustained and stable due to the integration of the inserted gene into the host cell genome and division as the cell genome divides. In addition, lentivirus has a wide range of hosts, can infect dividing and non-dividing cells, and can infect almost all types of cells, especially for cells with low transfection efficiency of plasmid vectors. [0003] At present, most of the commonly used lentiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/67C12N15/65C12N15/64C12N15/12
CPCC12N15/86C12N15/65C07K14/4702C12N2740/15043C12N2800/107
Inventor 陈英郭学双蒋桂梅李欣吴向萍赵政
Owner SHANDONG VIGENE BIOSCI
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