Experimental methods summary (1): gene manipulation tools
1. Construction of a GeneA knockdown vector
2. Construction of a GeneA overexpression vector
3. Construction of a GeneA point-mutation vector
4. GeneA knockout vector (CRISPR/Cas9)
5. GeneA knockout vector (TALENs)
6. GeneA miRNA expression vector
Version 1: For GeneA, search the Sigma website for shRNA sequences with validated knockdown efficiency. Add restriction sites to both ends and synthesize double-stranded DNA oligos with the corresponding sticky ends. Digest pEGFP with EcoRI and BamHI to linearize it, insert the shRNA expression cassette into pGP, transform competent E. coli, and identify positive clones by PCR and sequencing to construct an shRNA backbone plasmid targeting GeneA. The source names both pEGFP and pGP; verify the intended vector.
Version 2: Retrieve the human GeneA nucleotide sequence from GenBank. Design candidate GeneA shRNAs with Ambion software, assess homology using BLAST, select three more-specific candidates and use a replaceable stem-loop sequence as a negative control. Digest 15 μL pEGFP shRNA vector with 1.5 μL BamHI, 1.5 μL EcoRII and 5 μL 10× buffer for at least 4 h. Recover the product, ligate the GeneA shRNA fragment with T4 DNA ligase, transform DH5α competent cells, extract plasmids and identify positive clones by PCR and sequencing. EcoRII is reproduced as written; verify the enzyme and restriction sites.
Retrieve the target CDS from PubMed Nucleotide and analyze restriction sites using DNAMAN. Select EcoRI and SacII sites present in the cloning vector’s multiple-cloning site and absent from the target sequence. Design primers at the start and end of the GeneA CDS and add the sites at the 5′ ends. Amplify from cDNA, gel-purify the target, digest and purify it; similarly digest and recover pCDH vector. Ligate, transform DH5α competent cells, and identify positive clones by PCR and sequencing.
Version 1: Design complementary mutation-specific oligonucleotide primers. Amplify the plasmid with PrimeSTAR GXL DNA polymerase, add DpnI and incubate at 37°C for 1 h to digest methylated parental template, leaving the unmethylated mutant plasmid. Transform competent E. coli; the source states that plasmid nicks are repaired in the cells, yielding colonies containing the mutant plasmid.
Version 2: Use the Hieff Mut site-directed mutagenesis kit to construct a GeneA point-mutant vector. Design mutation-bearing primers, amplify the plasmid by inverse PCR, and digest methylated template with DpnI at 37°C for 1–2 h. Prepare the Exnase recombination reaction, incubate at 37°C for 30 min and cool in an ice-water bath for 5 min. Transform 200 μL competent cells with 20 μL cooled reaction, plate 100 μL on suitable antibiotic medium, and identify positive clones by PCR and sequencing.
Retrieve the GeneA CDS from NCBI, identify the exon to target and enter an exon sequence of 23–250 bp into sgRNA-design software. Synthesize complementary DNA oligos from the selected sgRNA sequence, anneal by gradual cooling, ligate into a Cas9 plasmid and transform DH5α cells. Sequence positive clones and prepare plasmid. The source refers to an shRNA expression element in the Cas9 plasmid; verify whether sgRNA was intended. For a mismatch-nuclease assay, PCR-amplify the target, denature and reanneal to form mismatched heteroduplexes, digest with CEL1 or T7E1, and compare cleaved and uncleaved bands by electrophoresis.
TAL effectors (TALEs) bind specific sequences; fusion to FokI creates TALEN genome-editing tools. First, identify candidate TALE sites in the selected target sequence, preferably with T as the first 5′ base. The historical source listed these online tools: https://boglab.plp.iastate.edu; http://idtale.kaust.edu.sa; http://zifit.partners.org/ZiFiT; and http://bloglabx.plp.iastae.edu/TALENT/help.php. These addresses are reproduced as written and have not been verified.
Second, obtain target-specific TALE proteins; the source refers to published improved methods [18–22]. Third, choose a FokI domain and assemble TALE and FokI into complete TALEN proteins. Fourth, introduce TALEN into cells for targeted genome editing. Fifth, screen positive clones and assess activity. The source notes that some workflows add an activity assessment such as yeast two-hybrid testing between steps three and four.
Experimental method:
Following the stated TALEN target-selection principles, design a target pair in the first exon of GeneA. Construct TALE-repeat vectors for the left and right half-sites and join them to pCS2. Transform the TALE module plasmids into competent TOP E. coli, grow cultures and extract plasmids using a Tiangen miniprep kit. Perform restriction digests with SpeI, NheI and HindIII, recovery, transformation and ligation; screen by colony PCR. Repeat digestion, gel recovery and ligation until both repeat arrays are complete, sequencing after each ligation round. Join the repeats to pCS2 and check inserts by digestion and SP6 sequencing.
Retrieve the GeneA mRNA sequence from GenBank. The historical source lists www．invitrogen.com/mai for miRNA design; verify availability and spelling. Select 21 nt target regions in GeneA mRNA. For the linearized pcDNATM6.2-GW/EmGFPmiR vector, design four 64 nt pre-miRNA oligos targeting different GeneA regions and their complements; use NCBI BLAST to assess nonspecific homology. The stated structure is 5′-TGC TG overhang + G + antisense target sequence + miRNA loop + sense target sequence-3′. Anneal four oligo pairs, ligate the double-stranded oligos into digested linear vector with T4 DNA ligase, transform DH5α, and screen clones by PCR, gel electrophoresis and sequencing.
Note: adapt the red text to the actual experimental conditions.
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