Small RNA virus-based expression vectors
Small RNA virus-based expression vectors
Small R N A viruses comprise a large group of small, pod-less, positive-sense single-stranded R N A viruses. Small R N A viruses have a rapid life cycle that takes place entirely within the cytoplasm without integrating into the host cell genome or undergoing a nuclear phase. Their use for usual gene transfer applications is limited by their biological and genetic limitations. However, they may be suitable as vaccine vectors. In addition, the small RNA virus-mediated expression of a variety of reporter genes or foreign RNA elements (foreign RNA d e m e n t ) is of great interest to small RNA virus molecular virologists. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Recombinant Small RN A Virus Genome-Directed Human Cells Move Recombinant Small RN A Virus Genome-Directed Human Cells Material reagents Agarose Calf serum Diethylaminoethylglucose (DEAE-dextran) (0.5m g/m l; M r= 500 000; Sigma-A ld H c h ) Add 20 mg of diethylaminoethyl dextran to 40 ml of HBSS in a sterilized Falcontube and shake vigorously for 5 min; store at 4°C for 1 month. D NASE (without R NASE, Roche 10776785001) D M E M (without serum, Invitrogen 11965-092) Different cell types may require other media. Ethanol [ 70 % (V /V) and 1 0 0 % ] H ank Equilibrium Salt Solution (H B S S ; Invitrogen 14025-092) H e L a R 19 cells (logarithmic growth phase) H 2O (ultra-pure water) The use of ultrapure water and chemicals for RN A eliminates the need to sterilize the solution under high pressure or to treat it with DEPC (diethyl pyrocarbonate). In vitro T7RNA polymerase kit (Stratagene 600124, or equivalent) Phenol: Chloroform [50:50 (V/V) Plasmid Plasmids should be constructed by inserting a recombinant virus or a replicating genome expressing the desired gene behind a T7 RNA polymerase promoter. Appropriate restriction endonucleases for plasmid linearization. T A E Buffer 40m m ol/L T r is lmmol/L EDTA 20 mmol/L acetic acid Instrument CentriSpin 20 columns (Princeton Separations, CS--2 0 l or equivalent) E P tubes Freezer 1 80℃ Incubator (37°C, CO2) Centrifuge shaker Cell culture dish (35m m diameter) Methods Transcription of recombinant R N A A 50ul transcription reaction should produce 5-20ug of RNA, enough to transfect several 35m m dishes. The approximate amount of RNA per 35m m dish is 2ug. If more dishes need to be transfected, increase the amount of transcription accordingly. 1. Linearize 1~2ug of virus or replicon plasmid containing the target gene. 2. Transcribe the linear template in vitro using a commercially available T 7 R N A Polymerase Kit with a final reaction volume of 50ul, see product description. After transcription, the template DNA can be removed by adding 10 units of DNase (without RNase) and incubating at 37℃ for 15m in, this step can be omitted. 3- Determine the RNA concentration. 4-Purify R N A in C entriSpin 20 c o lu m n (or similar desalting centrifugal column), see product description. Alternatively, it can be extracted with phenol:gasimide and washed with ethanol. If the concentration of RNA transcript is already high enough (>200 ng/ml), RNA purification can be omitted. 5 - Estimate the quality and integrity of the RNA by 1 % agarose gel electrophoresis using TAB buffer. 6. Use the RNA for transfection immediately or refrigerate at 80°C for later use. RNA Transfected Cells 7. 24 h before R N A transfection, H e L a R 19 cells in logarithmic growth phase were inoculated in 3 5 mm dishes. At the time of transfection, the cells should reach 50% to 70% confluence. 8-For each dish that will be transfected, mix 2F 1 in vitro transcription R N A (obtained in step 6) and 300ul diethylaminoethyl dextran (D E A E -dextran). Incubate on ice for 30m i n . 9 . Before transfection, wash HELA monolayer cells twice with cold HBS. 1 0 . Aspirate H B S S from the dishes and add 300ul of DEAE-dextran/RNA mixture to each dish. Place on a shaker and shake at room temperature for 30m i n . A control dish, i.e., without RNA, should be added; otherwise, the procedure is the same. Ensure that the cells do not dry out during the experiment. 11. Add 2 ml of pre-warmed DMEM (without serum) to each dish. Incubate at 37°C in a CO2 incubator for 1h. 12 - Aspirate the medium and add pre-warmed DMEM (with 2 % calf serum). Re-incubate at 37°C in a CO2-covered incubator. 13. Periodically check dishes for cytopathic effects (CPE) or for expression of inserted genes (replicons or recombinant viruses). The protein expression of subgenomic PV replicons undergoes a maximum of 6 to 12 h of post-transfection. Since no infectious particles are synthesized, no other cells are infected. As the transfected cells die, the expression decreases rapidly. The performance of cells infected by the viral constructs is similar in the first few hours of post-transfection. However, the infectious particles multiply and spread, killing all cells in the dish. In the wild type of PVNA, complete CPE can be observed within 24 h of post-infection. Virus-based expression vectors often have a characteristic delayed CPE (up to 4 d later). In general, the delay in the appearance of CPE compared to wild-type viruses represents a decrease in fitness of structures and genetic stability. 14. Repeated freezing and thawing of petri dishes 3 times causes the virus to be released from the cells. 15. Transfer the resulting liquid to an Ep tube and centrifuge at 14 OOOr/m i n for l m i n . 16. Store the virus-containing supernatant at 80°C. Viruses can be amplified by primary and secondary passages. However, because of the instability of most virus constructs, in subsequent experiments it is preferable to use the earliest available virus, and often new viruses are obtained by RNA transfection rather than by transmission. The use of oligonucleotide primers to amplify the insert fragments by reverse transcription PCR technology can monitor the stability of the virus constructs. For more product details, please visit Aladdin Scientific website.
Do not add more than 10ul of RN A per 300ul of DEAE-dextran. If the RNA concentration is already higher than ZOOng/ul after in vitro transcription, it can be added directly without purification and step 4 can be omitted.