Transposon-mediated delivery of small interfering RNAs: the "Sleeping Beauty" transposon experiment
Transposon-mediated delivery of small interfering RNAs: the "Sleeping Beauty" transposon experiment
A method for efficient delivery of pol III-driven SHRNA expression frames using the Sleeping Beauty (SB) transposon. Advantages of this system include its ease of gene delivery with standard plasmid transfection reagents and the increased efficiency of genome integration due to the possibility of gene translocation and multiple integration, which enhances s_A expression. Since this method requires only the transfection of plasmid DNA, there is no requirement for X# virus packaging and transduction. Author: T. Friedman et al, Translator: Jingwei et al, This experiment is from "Gene Transfer".
Operation method
Transposers s iR N A Transportation Move Transposers s iR N A Transportation Material reagents Agarose gel (0.9 %), prepared according to the standard method. 9 %), prepared according to standardized method B G H reverse primer (5'-T A G A G A G G C A C A G TC G A G G --3. (In vitrogen ) Transfection reagent LIP ofectAMINE Plus (Invitrogen) NEBuffer 3 (N e w England Biolabs) Code Targeting Oligonucleotides of gene s h R N A High-performance liquid chromatography (HPLC) purification with 5'-end pittyration is recommended (OperonBiotechnologies, Huntsville, Alabama). Q I A E X II Gel Extraction Kit (Q I A G E N ) Q I A G E N Ma x i Prep Restricted enzymes: BwBI and EcoRI (England Biolabs) SB transposon plasmid containing a polIII promoter that drives SHRNA expression pMaleficent (Heggestad et al. 200 4 ) or the FP system pFP/Neo^Hl (Kaufman et al. 2005). SB transposase expression plasmid pCM V-SBlO (Ivies et al. 1997). Other high-activity subversion mutants can also be used, such as pCMV-HSB17 (Bausetail.2005), pCMV-HSB4 (YantetaL 2004). If FP is used, the expression plasmid PFV-FP (Kaufman et al. 2005) can be used. Culture medium containing G 418 (0.5~l m g /m l ) T 4 D N A ligase (N e w England Biolabs) Target cell lines for transient transfection Instrumentation Boiling water bath Tissue Culture Plate (IO cm) 16°C, 37°C and 55°C water baths Methods Synthesis of oligonucleotides 1 . Design Oligonucleotide pairs for target genes 2- For the pMaleficent carrier, the overhangs at the 5' and 3' ends are designed to be compatible with the B s m B I and E c 0R I restriction linkage sites and to contain the appropriate loop structure and termination signals (H e g g e s t a d e t a L 2004). For the F P carrier p F P /N e o -H l , consider Bglll and HindIII. 3- Oligonucleotide pairs (each 50 p m o l dissolved in 50 Ul of distilled water) were reconstituted in a boiling water bath for 2 m i n , and slowly cooled to room temperature (about 20 m i n over). Attachment to transposon 4.37. 3 7 . The transposon plasmid pMaleficent(2ug)lh was sheared with approximately 20U of Ec:oRI in NEBuffer3 at 4 .3 7 C (reaction volume 20ul). 5 . Add IOU Bsm BI to the above reaction tube and incubate for 2 h at 55°C. 6. Separate the digest with 0-9 % agarose gel and purify the approximately 6 kb of sheared tape with QIA EX II Gel E x tractio n K it. 7 . In a 20 M1 reaction, the agarose gel-purified carrier (1 ul or 50 ng) is attached to the reprocessed oligonucleotide (5 ul) with T4 DNA linker enzyme. 8 . Bacterial transfection of the ligated product (approx. 3ul), ampicillin-resistant colonies were picked and analyzed with the BGH reverse primer V -TA G A G A G G C A C A G T C G A G G G G ^ . 9- After identifying the correct clone, the plasmid was amplified in large quantities (Q I A E N Maxi P rep ). Cell transfection 10 . High-quality plasmid DNA is transfected into target cells using the transfection reagent LipofectA M IN E P l u s or other high-transfection methods. 11. A mass ratio of 4:1 is recommended for pM aleficen t and transposase-expressing plasmids. 12- After 2d of transfection, the cells were collected and counted. The cells were collected and counted. 5 X 10 The number of cells grown on the culture plates depends on the transfection efficiency and the activity of intracellular SB translocation. 13 . Collect tolerogenic clones, or isolate individual tolerogenic clones. Cells are frozen and analyzed for down-regulation of gene expression by protein tracing or other methods. For more product details, please visit Aladdin Scientific website.
Sequence design methods are described in the literature (Schwarz et al. 2003; Reynolds et al. 2004; Boese et al. 2005; Heale et al. 2005). Sequence design methods are also available from OligoEngine (littp://www.cerebre^com/indeundefinedhtml) or Ambion
(h ttp ://www.ambion.com/techlib/tb/tb _ 506. html) for online help. Synthesize at least three oligonucleotides with different sequences and a control oligonucleotide pair.
3 to IXIO5 cells were planted on an IOcm culture plate and cultured in a medium containing G 418 (0.5~l m g/ml) for 14d, and the medium was changed every 7 days.