Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

siRNA, kit and application for knocking down porcine samhd1 gene expression

A gene expression and kit technology, applied in the fields of molecular biology and cell biology preparation, can solve the problems of not finding knockdown genes, limited lentivirus infection efficiency, limitations, etc., achieving high knockdown efficiency and broad application prospects. , the effect of improving infection efficiency

Active Publication Date: 2022-04-22
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI +1
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Limited by the infection efficiency of lentivirus, the application of lentiviral vector is limited
In addition, no knock-down genes that can improve the infection efficiency of different cells have been found so far.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • siRNA, kit and application for knocking down porcine samhd1 gene expression
  • siRNA, kit and application for knocking down porcine samhd1 gene expression
  • siRNA, kit and application for knocking down porcine samhd1 gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example one pig SAMHD1 gene knockdown inefficiency detection

[0032] 1.1. siRNA design

[0033] According to the porcine SAMHD1 gene sequence (NM_001292105.1) published in the NCBI database, 5 pairs of siRNA combinations were designed, recorded as siRNA-1, siRNA-2, siRNA-3, siRNA-4, siRNA-5, synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd., and the sequence is shown in Table 1.

[0034] Table 1 siRNA designed based on the SWHD1 gene in pigs

[0035]

[0036]

[0037] 1.2. siRNA transfection

[0038] When the IPAM cell density is grown to 80%, the siRNA is transfected to IPAM cells using the jetPRIME Polyplus Transfection Kit (Ref #114-15 / 1.5 mL), and the final concentration of transfection is blank (NC), 50 nM / μL, 100 nM / μL, 150 nM / μL, and 200 nM / μL, respectively. Replace the medium with an equal volume of serum-free RPMI1640 medium 4-6 h after siRNA transfection.

[0039] 1.3. Lentiviral packaging

[0040] 1.3.1 Calculation of transfection volume of e...

Embodiment 2

[0076] Example 2 measures the efficiency of lentiviral infection

[0077] 2.1. Lentiviral packaging

[0078] See Example 1.3.

[0079] 2.2. Transfection and grouping of siRNA to 1 mL volume per well to 2×10 5 / mL density is placed in 12-well cell culture plates, with a total of 12 mL used for 1 plate. When the cell density is grown to about 80%, the siRNA provided by the kit is transfected into 6 of the cells at a final concentration of 150 nm / μL using the jetPRIME PolyplusTransfection Kit (Ref #114-15 / 1.5 mL) as the experimental group; the other 6 wells are transfected with the same concentration of NC-siRNA as the control group. Each group of cells was replaced with serum-free RPMI 1640 medium 4 h after siRNA transfection.

[0080] 2.3. Lentiviral virus infection and grouping

[0081] Transfected 6-well cells of siRNA provided by the kit, Lenti-gfp was added to 3 of the well cells at a final concentration of MOI= 100, which was denoted as S1; the other 3 well cells were not trea...

Embodiment 3

[0091] Example three lentivirus infection BMDM cells

[0092] The experimental procedure refers to Example II infection of BMDM cells. Lentivirus-gfp with MOI = 1000 poisoned each well BMDM cell flow cytometry cell population results as shown Figure 16 as shown. According to statistical analysis, the infection efficiency of BMDM cells at the moi=100 infection level also reached 25%, which was significantly different from the control.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the technical field of molecular biology and cell biology preparation, and discloses a siRNA for knocking down the expression of porcine SAMHD1 gene, a kit and its application. The positive-sense strand of the siRNA is shown in SEQ ID NO.1 Base sequence, the antisense strand of the siRNA is the base sequence shown in SEQ ID NO.2. The siRNA used for knocking down the expression of porcine SAMHD1 gene in the present invention has high knockdown efficiency, is suitable for the experimental operation of pig-derived cell infection using lentivirus as a tool, can significantly improve the efficiency of lentivirus infection of pig-derived cells, and has broad application prospects .

Description

Technical field [0001] The present invention belongs to the field of molecular biology and cell biology preparation technology, relates to a siRNA for knocking down the expression of the SAMHD1 gene in pigs, a kit and its application. Background [0002] Lentivirus belongs to the retroviridae family, more than macrophages, lymphocytes in the primary infection, after a long incubation period to make individuals show obvious clinical symptoms, because of its characteristics, such viruses are called lentivirus. The commonly used lentiviral vector is a human immunodeficiency type I virus (HIV-1), which has the advantages of widespread infection, effective infection of division and quiescent phase cells, insertion of large genetic fragments in the host chromatin, and long-term maintenance of stable transgene expression, so it has become a favorable tool for the introduction of foreign genes. Limited by the efficiency of lentiviral infection, the application of lentiviral vectors is l...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/55C12N15/867A01K67/027
CPCC12N15/113C12N9/80C12N15/86A01K67/0276C12N2740/15041C12N2740/15052A01K2227/108
Inventor 王琦曹丽艳袁聪段月月索学鹏孔祥雨
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com