Cationic polysaccharide assay for DNA delivery

Summary

Polycations are an effective class of non-viral gene delivery vectors. These vectors vary in molecular mass, chemical structure, polymer/DNA ratio, and molecular structure, and possess the ability to bond targeting groups. These vectors can be complexed with different plasmids and transfected into a variety of cells for efficient expression of target proteins. Cationic polysaccharides are a class of vectors of great interest in gene delivery. These polysaccharides are derived from natural or semi-natural materials, are non-toxic, biodegradable, have good biocompatibility, and can be easily modified to improve their physicochemical properties. Author: T. Friedman et al., Translated by W. Qin et al. This experiment is from "Gene Transfer".

Operation method

Synthesis and in vitro transfection of cationic polysaccharides

Move

Synthesis and in vitro transfection of cationic polysaccharides Materials

reagents

Bicinchoninic Acid (BCA) kit (Pierce)

(3-gal ELISA (Roche) or (3-gal Assay (Invitrogen))

Calcium phosphate reagent (Sigma-Aldrich)

Cells to be transfected, e.g.

C 3 H 10T 1/2

H E K 293

C H O

H eL a

N IH 3T 3

EPC

COS-7

Dextran (mean molecular mass 40kD a) (Sigma-Aldrich )

Fetal Bovine Serum (FBS) (Beit Haemek, Israel)

GLUTAMINE (4nm ol/L) (B eit H aem ek, Israel)

HEPES■ buffered saline (HBS; 150 mmol/L NaCl, 20 nmol/L HEPES, pH 7.1)

Sterilize by filtration with 0.2um membrane or autoclave at 4°C.

Human Growth Hormone (hGH) ELISA Kit (Roche)

Hydroxylamine hydrochloride

Phospholipid Formulation

D O T A P /C h o l 1/1 (A vanti P o lar Lipids Inc., A labam a)

T ra n sfa st (P rom ega)

F u g e n e 6 (R oche)

Fluorokinase Reporter Gene Assay (R o c h e ) or Fluorokinase Assay Kit (P r o m e g a )

Culture medium

Complete medium: DMEM with 10% (m/m) fetal bovine serum (FBS), 0.2 mmoI/L L-glutamine, lmg/m l penicillin, IOOUAnl streptomycin
D M E M (D ulbecco's m odified E agle's m edium )

Nitrogen (for storage of complexes)

Penicillin (Beit H aem ek, Israel)

Plasmid

pEGFP-Cl, CM V promoter; pLN Cluc Plasmid, containing the firefly fluorophore enzyme gene; pLacZ, SV4 0 or CM V promoter; pCM VhGH for quantitative and qualitative analysis. Plasmids were purified using a Nucleobond AX 500 column (Machery-Nagel, Duren, Germany) or a QIAGEN Plasmid Mega Kit (Hilden, Gerimany). pLN Cluc plasmid contains the firefly fluorophore enzyme, pLacZ, SV4 0 or CM V promoter, and pCM VhGH was used for quantification and characterization.

Potassium iodate (K I O 4 ) (Sigma-Aldrich)

Sodium Borohydride (N a B H 4) (Sigma-Aldrich)

Arginine amine (Sigma-Aldrich)

Streptomycin (Beit Haemek, Israel)

Instrumentation

Cellulose dialysis tubing (retained molecular mass 3500)

6-well cell culture plates

D o w e x - I (Acetate) Cation Exchange Resin

Sudden light microscope (M o d e l Axiovert 35, Zeiss, Jena, G e r m a n y )

Lyophilizer

Spectrophotometer, N M R (Nuclear Magnetic Resonance Spectrometer), Infrared

Methods

Synthesis of cationic polysaccharides: oxidation of dextran

1- Dissolve dextran (l0 g, 62.5 m m l of sugar units) in 200 m l of secondary deionized water.

2. Add potassium periodate at a molar ratio of I : 1 (sugar _unit/IO4- ) and stir for 6~8 h at room temperature away from light.

3 . Purify the polyaldehyde by removing the IOr and unreacted IOr through a D owex-1 ion exchange column. The polyaldehyde was purified by removing the IOr and unreacted IOr on a D owex-1 ion exchange column and analyzing with secondary deionized water at 4°C (cellulose dialysis tubing, 3500 retention mass) for 3d.

4 . Lyophilized purified polyaldehyde derivative. The product is a white powder.

The average yield was 7 0 %. The C= O absorption peak was observed at 1724 cm-1 by FT-IR (KBr).

5. The polyaldehyde content was determined by titration with hydroxylamine hydrochloride (Zhao and Heindel 1991).
The oligoamine bonding

6 - Dissolve the oxidized dextran (l g, 0.75 to 6.56 m m o l aldehyde group) in IOO m l of secondary deionized water. It was added slowly dropwise to 50 ml of borate buffer solution containing 50 % excess spermine.

7 . Stir for 24 h at room temperature. Reduce the imine to the amine by addition of N a B H 4 (l g, 4 times the aldehyde molar number). Continue stirring for 48 h.

8- Repeat the reduction for 24 h with additional NaBH 4 (l g, 4 times the aldehyde molarity).

9-Transfer the bright yellow solution to dialysis tubing (cellulose dialysis tubing with a 3500 retention mass) and analyze for 3d at 4°C with secondary deionized water.

1 0 - Lyophilized dialysis product, stored under nitrogen. The average yield was 5 0% (OT/m ). The amino ratio of the product was obtained by NMR analysis of the product. (m ) multiple peaks, (H ) protonated hydrogen.

The transfection efficiency (transfection percentage) was calculated by dividing the number of cells by the total number of cells in the field of view.
In some cases, gene expression can be assayed for total amount of protein using standard BCA kits, normalizing the results to

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