基于mir-30结构的shRNA的构建（以pINDUCER10）为例

                                              在pINDUCER10中构建USP2敲减质粒

一、pINDUER10(miR-RUP)的质粒图谱

Addgene linker：http://www.addgene.org/44011/

二、USP2 shRNA设计

根据以前设计shRNA经验，在Thermofisher网站上设计得到五星的siRNA序列AGATTGTGGTTACTGTTCT(5’→3’)，接下来按照如下操作进行

1.      Convert the predicted 21-nucleotide siRNA guide sequence to a 22-nucleotide guide sequence.
This step is critical because miR-30-based vectors generate 22-nucleotide rather than 21-nucleotide guide and passenger strands.
i. Begin with a predicted (e.g., predicted by DSIR, Biopred, or another algorithm, or found in the literature) 21-nucleotide antisense guide sequence. USP2 sense siRNA的为CAGATTGTGGTTACTGTTCTA
ii. Reverse-complement this sequence to find the target site on the mRNA and verify that the predicted guide targets the mRNA of interest.
iii. Extend the target site by appending the base immediately 5′ on the mRNA to generate the 22-nucleotide target.
iv. Reverse-complement this to produce the guide strand of the shRNA.（在USP2 siRNA5‘端再加一位A）

2.       The miR-30-based shRNAs retain a bulge（突出部分）at the base of the shRNA stem that is predicted to exist in the bona fide miR-30 primary transcript. This impacts the design of the sense strand. Beginningwith the target site derived above, alter the sense strand as follows. If the first base is A or T, convert it to C. If the first base is C or G, convert it to A. This generates the appropriate bulge in the final, folded RNA.（首位A变为C，因此sense siRNA为CCAGATTGTGGTTACTGTTCTA）

3.       The oligonucleotides for annealing and cloning are designed by adding flanking sequence derived from the miR-30 pri-miRNA and sequences compatible with restriction sites. For miR-30-based vectors that use XhoI and EcoR1 as cloning sites, create sequences of oligonucleotides according to the following formulas.
i. Top strand: Insert the gene-specific 22-nucleotide sense and 22-nucleotide antisense sequences into the following pattern and chemically synthesize the resulting 110-base oligonucleotide.
5′-[TCGA]GAAGGTATATTGCTGTTGACAGTGAGCG + sense 22-mer + TAGTGAAGCCACAGATGTA (mir30 loop) + antisense 22-mer + TGCCTACTGCCTCGG-3′
ii. Bottom strand: Remove the bracketed TCGA, which formed the overhang compatible with XhoI. Reverse-complement the sequence and append AATT at its 5′ end to create an overhang compatible with EcoR1. Synthesize the 110-base oligonucleotide. The annealed oligonucleotides will have the following structure (X denotes a variable position):
5′-TCGAGAAGGTATATTGCTGTTGACAGTGAGCGXXXXXXXXXXXXXXXXXXXXXXTAGTGAAGCCACAGATGTAXXXXXXXXXXXXXXXXXXXXXXTGCCTACTGCCTCGG-3′
3′-CTTCCATATAACGACAACTGTCACTCGCXXXXXXXXXXXXXXXXXXXXXXATCACTTCGGTGTCTACATXXXXXXXXXXXXXXXXXXXXXXACGGATGACGGAGCCTTAA-5′

根据以上方式，设计USP2 shRNA如下：

5′-（Xhol I酶切位点）TCGA GAAGGTATATTGCTGTTGACAGTGAGCG CCAGATTGTGGTTACTGTTCTA TAGTGAAGCCACAGATGTA TAGAACAGTAACCACAATCTGG TGCCTACTGCCTCGG-3′
3′-CTTCCATATAACGACAACTGTCACTCGC GGTCTAACACCAATGACAAGAT ATCACTTCGGTGTCTACAT ATCTTGTCATTGGTGTTAGACC ACGGATGACGGAGCC（EcoR I酶切位点）TTAA -5′

酶切位点选Xhol I和EcorI，且已验证shRNA序列中不存在其酶切位点
iii. Order the designed oligonucleotides from an  appropriate supplier. Once the ordered oligonucleotides arrive, anneal and ligate them into the vector as described in the following steps.

 4. Begin by reconstituting each olignucleotide in H2O to identical concentrations (0.5 µg/mL is
convenient).
5. Set up the annealing reaction by mixing the following reagents in a PCR tube:

Top strand     20 µL    

Bottom strand     20 µL    

Annealing buffer (5×)     10 µL    

6. Execute the following program in a PCR machine:
5 min at 95˚C
10 min at 80˚C
5–7 h ramp from 80˚C to 4˚C (–0.5˚C every 2.5 min)
7. Dilute 10 μL of annealed oligonucleotides 1:10 with ddH2O or EB buffer. Store the undiluted and
diluted shRNA inserts at –20˚C.
8. Ligate the diluted shRNA insert into a suitable vector that has been digested with EcoRI and XhoI
and purified (e.g., pSM2, pGIPZ, pTRIPZ, Open Biosystems) as follows:

XhoI/EcoRI-cut miR-30 vector (50–200 ng)     2 µL    

Diluted annealed insert     2 µL    

H2O 12 µL
T4 DNA ligase buffer (10×) 2 µL
ATP (10 mM) 1 µL
T4 DNA ligase 1 µL
Ligate for 2 h at 20˚C.
9. Use 3–5 µL of the ligation mixture from Step 8 to transform 50–100 µL of competent cells.
10. Pick three to five colonies, prepare plasmid DNA, and check by restriction digest and sequencing.