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
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.

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
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.

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.

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