Site directed mutagenesis

Manufactured by New England Biolabs

Site-directed mutagenesis is a technique used to make specific and controlled changes in the DNA sequence of a gene or plasmid. It allows for the introduction of desired mutations, deletions, or insertions at precise locations within a target DNA molecule.

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8 protocols using site directed mutagenesis

Characterization of PHM-2 Isoform Expression

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To analyze transcription of phm-2 isoform a (F32B4.4a), we PCR amplified the 496bp region between the start codon of phm-2 and the 3’ end of the adjacent upstream gene from genomic DNA of wild-type animals. This promoter region was inserted into plasmid pDG218 to generate pSK1 [phm-2p::gfp::unc-54 3’UTR]. Briefly, pDG218 was derived from pBluescript SK+ (Stratagene) by inserting the coding region for green fluorescent protein (GFP) and the unc-54 3′-UTR, both amplified from pPD95.77, a gift from A. Fire (Stanford University, Palo Alto, CA).
To analyze expression of PHM-2 protein, we PCR-amplified full length, wild-type phm-2 coding sequence (3222 bp) from cDNA and ligated into pSK1 to generate pSK2 [phm-2p::phm-2::stop codon::gfp::unc-54 3’UTR]. pSK2 encodes PHM-2 protein but the gfp coding sequence is out-of-frame following the stop codon of phm-2. The stop codon in pSK2 was removed by site directed mutagenesis (New England Biolabs) to generate pSK3 [phm-2p::phm-2::gfp::unc-54 3’UTR]. pSK3 encodes a PHM-2::GFP fusion protein.

Kumar S., Egan B.M., Kocsisova Z., Schneider D.L., Murphy J.T., Diwan A, & Kornfeld K. (2019). Lifespan extension in C. elegans caused by bacterial colonization of the intestine and subsequent activation of an innate immune response. Developmental cell, 49(1), 100-117.e6.

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Site-Directed Mutagenesis of miRNA Target Sites

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Primers were designed using the New England Biolabs Site-Directed Mutagenesis online design tool (https://nebasechanger.neb.com/; see Supplementary Table 2). The targeted sequence alterations designed to ablate the putative miR interactor sites were, for miR-21-5p, a 7-nt substitution with the complementary sequence, whereas for the remaining of miRs a 6–8 nt deletion spanning the interactor sites was introduced. Site-Directed Mutagenesis reactions were performed according to the PCR protocol (Q5 polymerase), using 20 ng of CDK2AP1–3’UTR luciferase reporter plasmid, supplemented with 5% DMSO in the case of the deletions. PCR products were digested with DpnI to eliminate the template DNA, and ligated in 1x KLD (Kinase-Ligase-DpnI) Reaction Buffer (Q5 Site-Directed Mutagenesis Kit, E0552S, New England Biolabs) for 30 min at room temperature. Chemically competent bacteria (Thermo Fisher, One Shot™ TOP10 Chemically Competent E. coli, C404010) were transformed according to the manufacturer’s protocol in 5 μl KLD reaction. Plasmid DNA was extracted from positive colonies and subsequently sequenced to ensure the correct product was obtained.

Stabile R., Cabezas M.R., Verhagen M.P., Tucci F.A., van den Bosch T.P., De Herdt M.J., van der Steen B., Nigg A.L., Chen M., Ivan C., Shimizu M., Koljenović S., Hardillo J.A., Verrijzer C.P., Baatenburg de Jong R.J., Calin G.A, & Fodde R. (2023). The deleted in oral cancer (DOC1 aka CDK2AP1) tumor suppressor gene is downregulated in oral squamous cell carcinoma by multiple microRNAs. Cell Death & Disease, 14(5), 337.

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Cloning and Modifying ZMYM Genes

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Human ZMYM family genes (ZMYM2, ZMYM3, ZMYM4 and ZMYM6) were amplified by PCR from HEK293T cDNAs. Amplified ZMYM gene products were cloned into a pDONR201 vector using Gateway cloning (Invitrogen) following the manufacturer's protocol. ZMYM1 in pENTR223 vector was purchased from Harvard plasmid (Clone ID:HsCD00365611). ZMYM5 in M29 vector was purchased from (GeneCopoeia, Inc., Cat No. EX-Z6750-M29). ZMYM cDNAs were then sub-cloned into pDEST mammalian expression vector as described previously (17 (link)). For ZMYM2, this gene was sub-cloned from pDONR201 into various Gateway destination vectors containing epitope tags as indicated. Deletion mutants of ZMYM2 were created in pDONR201 by PCR-amplifying ZMYM2 cDNA using forward and reverse primers containing complementary sequences to the regions flanking the area to be deleted. SUMO-interacting motif mutant forms of ZMYM2 were generated by site-directed mutagenesis (New England Biolabs) using primers that created alanine substitutions at the indicated amino acid positions.

Lee D., Apelt K., Lee S.O., Chan H.R., Luijsterburg M.S., Leung J.W, & Miller K.M. (2022). ZMYM2 restricts 53BP1 at DNA double-strand breaks to favor BRCA1 loading and homologous recombination. Nucleic Acids Research, 50(7), 3922-3943.

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Lentiviral Overexpression and CRISPR Editing

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The entry vector pDONR223 containing the full-length GRN (1–1179 base pairs) from the human orfeome collection was used to engineer a stop codon by site-directed mutagenesis (New England Biolabs) at the end of the open-reading frame sequence (ORF). Gateway technology (Thermo Fisher) was used to transfer the GRN ORF with LR cloning from the entry vector to the pHAGE lentiviral destination expression vector. sgRNA sequences for editing the TMEM192, GRN, HEXA, NPC1 and NPC2 loci were cloned into the pX459 V2.0 vector (Addgene Cat#62988) as described^{31 (link)}.

Boland S., Swarup S., Ambaw Y.A., Malia P.C., Richards R.C., Fischer A.W., Singh S., Aggarwal G., Spina S., Nana A.L., Grinberg L.T., Seeley W.W., Surma M.A., Klose C., Paulo J.A., Nguyen A.D., Harper J.W., Walther T.C, & Farese RV J.r. (2022). Deficiency of the frontotemporal dementia gene GRN results in gangliosidosis. Nature Communications, 13, 5924.

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Zebrafish ephrinb2a gene manipulation

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Zebrafish ephrinb2a (gene name efnb2a) was amplified from 2 dpf cDNA with the following primers: efnb2a forward (5′-ATCTCTAGAATGGGCGACTCTTTGTGGAGAT-3′) and reverse (5′-ATCAAGCTTGTTCATCGAGGGGCATGTGA-3′). The fragment was then inserted into pcDNA3.1(−) vector between XbaI and HindIII. A 4A mutant construct was generated using site-directed mutagenesis (New England Biolabs, Inc.) with the following primers: forward (5′-CAGAGCCCAGCAGCCGCCGCTGCCAAGGTGTGAAAA-3′) and reverse (5′-TGGCGGCATTTCCTGTACGAT-3′). Both ephrinb2a (WT) and ephrinb2a (4A) plasmids were digested by HindIII and then purified by column (Macherey-Nagel Inc.) as a template for in vitro transcription. Capped mRNA was produced with mMESSAGE mMACHINE T7 ULTRA Transcription Kit (AM1345; Thermo Fisher Scientific) and purified through lithium chloride precipitation described in the same kit. The concentration of mRNA was measured by NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific), and their quality was confirmed by electrophoresis in 1% (wt/vol) agarose gel. 500 pg of mRNA was injected into one-cell-stage embryos.

Li W., Wen L., Rathod B., Gingras A.C., Ley K, & Lee H.S. (2022). Kindlin2 enables EphB/ephrinB bi-directional signaling to support vascular development. Life Science Alliance, 6(3), e202201800.

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Site-Directed Mutagenesis of SIV Plasmids

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Nucleotide substitutions were introduced into the p239SpE3’:K180S hemiviral plasmid by site-directed mutagenesis (New England Biolabs). Mutated p239SpE3’:K180S hemiviral plasmids were digested with SphI-XhoI and joined with the 5’ half of SIV_mac239 SpX and SIV_mac239 SpX ΔVif. Reconstructed full-length infectious molecular clones were sequence confirmed by the UW-Madison Biotechnology Center. Proviral DNA without the ΔVif mutation was transfected into HEK293T cells using GenJet (SignaGen) to generate viruses for neutralization assays. SIV_mac239 SpX ΔVif plasmids were co-transfected with VSV-G envelope (pHDM.NJ strain) in a 2:1 ratio to generate viruses for ADCC assays and flow cytometry analyses. Culture supernatants were collected 48 h post-transfection, cleared of cell debris by centrifugation and concentrated on 50K MWCO centrifugal filters (Millipore Sigma). Concentrated viruses were well-mixed, aliquoted, and stored at -80°C. Concentrations were determined by anti-p27 ELISA (ABL, Inc.).

Grunst M.W., Gil H.M., Grandea AG I.I.I., Snow B.J., Andrabi R., Nedellec R., Burton I., Clark N.M., Janaka S.K., Keles N.K., Moriarty R.V., Weiler A.M., Capuano S I.I.I., Fennessey C.M., Friedrich T.C., O’Connor S.L., O’Connor D.H., Broman A.T., Keele B.F., Lifson J.D., Hangartner L., Burton D.R, & Evans D.T. (2024). Potent antibody-dependent cellular cytotoxicity of a V2-specific antibody is not sufficient for protection of macaques against SIV challenge. PLOS Pathogens, 20(1), e1011819.

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Peptide Synthesis and pMHC Refolding

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Peptides used for pMHC refolding were synthesized in house using the Liberty Blue Peptide Synthesizer (CEM) and validated using liquid chromatography–mass spectrometry (Agilent). To generate pMHC molecules for bioconjugation, codon-optimized gBlocks for H2-D^b and H2-K^b β2m were purchased from Integrated DNA Technologies and cloned into pET3a vectors (Novagen). H2-D^b and H2-K^b genes were engineered with a C-terminal cysteine by site-directed mutagenesis (New England Biolabs), and pMHC molecules were expressed and refolded as described previously (23 (link)).

Su F.Y., Zhao Q.H., Dahotre S.N., Gamboa L., Bawage S.S., Silva Trenkle A.D., Zamat A., Phuengkham H., Ahmed R., Santangelo P.J, & Kwong G.A. (2022). In vivo mRNA delivery to virus-specific T cells by light-induced ligand exchange of MHC class I antigen-presenting nanoparticles. Science Advances, 8(8), eabm7950.

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Genetic Manipulation and Hypoxia-Reoxygenation Assay

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HEK293 cells were cultured in high glucose DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin–streptomycin (Gibco). HA-RIPK1 and point mutant plasmids of HA-RIPK1 (K3R, K115R, K163R, K316R, K571R, K604R, and K627R) were gifts from Jaewhan Song (obtained from Addgene). MG53 C14A and HA-RIPK1 double mutant plasmids were generated by site-directed mutagenesis (New England Biolabs). HA-RIPK1 triple mutant plasmid was generated by the Mutagenex Company. The day before plasmids transfection, HEK293 cells will passage to new plates. After transfection, the cells will be collected for further experiments.
Human-induced pluripotent stem cells (hiPSC) were purchased from Gibco (A18945) and kept in the Essential 8 basal medium (Gibco). The cardiomyocyte differentiation kit (Gibco) was used for cardiomyocyte differentiation following our published protocol (22 (link)). After cardiomyocyte differentiation, 0.5% oxygen in nitrogen gas (99.5%) was used for hypoxia and the re-oxygen (H/R) treatment. The re-oxygenation condition was the same as the culture condition (95% air and 5% CO₂). Recombinant protein MG53 was added to the medium before hypoxia treatment.
CRISPR/Cas 9-mediated mg53-/- cell and wild-type parental cells were also used for H/R treatment.

Wang Q., Park K.H., Geng B., Chen P., Yang C., Jiang Q., Yi F., Tan T., Zhou X., Bian Z., Ma J, & Zhu H. (2022). MG53 Inhibits Necroptosis Through Ubiquitination-Dependent RIPK1 Degradation for Cardiac Protection Following Ischemia/Reperfusion Injury. Frontiers in Cardiovascular Medicine, 9, 868632.

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