Zymopure 2 plasmid maxiprep kit

The 14-kb plasmid harboring the lng operon of the CS21 pilus was purified from the ETEC E9034A∆lngA strain [20 (link)] using a ZymoPURE™ II Plasmid Maxiprep Kit (Zymo Research, Irvine, CA, USA). The lngA gene was previously replaced with a kanamycin cassette using the one-step inactivation method [23 (link)]. Mobilization of the plasmid into ECBL was performed via electroporation (BMC Harvard Apparatus, Cambridge, MA, USA) at 1800 V, and the transformed strains were selected by overnight culture on LB agar plates with kanamycin. The positive colonies were named ECBLpE9034AΔlngA (ECBLΔlngA). The DNA product of the ECBLΔlngA strain was confirmed by PCR using specific primers for the lngA, lngD, and lngH genes (Table 3) and by sequencing. In addition, electrocompetent cells derived from the ECBLΔlngA strain were transformed by electroporation with 100 ng of the pJET1.2 plasmid, which carried variants of the lngA gene of the clinical ETEC strains selected for this study (Table 4). The transformed colonies were considered positive when cultured on LB agar plates with both kanamycin and ampicillin.

Plasmids encoding the SARS-CoV-2 open reading frames proteins and eGFP control were a kind gift of Nevan Krogan (Addgene plasmid #141367–141395). Plasmids were acquired as bacterial LB–agar stabs and used per the provider’s instructions. Briefly, each stab was first seeded in LB agar (Bacto Agar; BD Biosciences, San Jose, CA) in 10 cm plates. Then, single colonies were inoculated into flasks containing LB (BD Difco LB Broth, Lennox) and 100 µg/ml penicillin (Biological Industries, Beit HaEmek, Israel). Transfection-grade plasmid DNA was isolated from each flask using the ZymoPURE II Plasmid Maxiprep Kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions.

We purchased the hemophilia A (HA) mice bearing an F8 exon 16 knockout on a 129 × B6 background from the Jackson Laboratory (Bar Harbor, ME), which was initially obtained from Dr. H. Kazazian (University of Pennsylvania) [20 (link)]. The mice were housed and maintained at the State Key Laboratory of Experimental Hematology (SKLEH, Tianjin, China). Animal experiments were conducted according to the protocols approved by the Institutional Animal Care and Use Committee of SKLEH and the Institute of Hematology. Vectors for hydrodynamic tail vein injection were prepared using the EndoFree MaxiPrep Kits (Qiagen) or ZymoPURE II Plasmid Maxiprep Kit (Zymo Research). We screened endotoxin using Lyophilized amebocyte lysate LAL/TAL reagent (Xiamen Bioendo Technology) and abandoned endotoxin-contaminated plasmids. Before in vivo injections, we diluted plasmid DNA using sodium lactate Ringer’s solution (China Otsuka Pharmaceutica). For hydrodynamic injection, a volume equivalent to 10% of mouse body weight was administered via the tail vein in 5–6 s into 5–8-week-old HA mice. The amount of plasmid DNA was 10 μg each for Cas9, sgRNA, and pDonor. To prevent bleeding, we injected 0.5 IU F8 protein (Xyntha; Wyeth Pharmaceuticals) in each mouse, together with the editing plasmids.

Plasmids pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene plasmid #62988) and pSpCas9(BB)-2A-GFP (PX458) (Addgene plasmid #48138) were a gift from Feng Zhang [59 (link)]. Endotoxin-free plasmids were extracted using ZymoPURE II Plasmid Maxiprep Kit from Zymo Research. Lipofectamine™3000, Texas-Red DHPE, and LysoSensor Green DND-189 were purchased from ThermoFisher Scientific. 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and paraformaldehyde and dimethyl sulfoxide (DMSO) were purchased from Sigma. 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Biotium. All cell culture reagents were purchased from Biochrom. Venor™ GeM Mycoplasma Detection Kit was purchased from Merck. The gene target sequences were synthesized by Alfagene. Lipids MVL5, DOTAP, DOPC, and DOPE were purchased from Avanti Polar Lipids (USA). GMO was purchased from Nu-Chek Prep (Elysian, MN, USA). All lipids were used as received. F-Luc-GFP lentivirus was purchased from Capital Biosciences.