ATM and Bnip3 double knockout and p53 and Bnip3 double knockout ESCs were generated using a CRISPR-Cas9 system. ATM and p53 gRNA were cloned into the px330 vector, and transfected into Bnip3 knockout ESCs with a Gene pulser Xcell II (Bio-Rad) according to the manufacturer’s protocol. Then, knockout ESCs were identified by digestion with PstI for ATM and NcoI for p53 and sequencing. All knockout ESCs were further identified by western blot.
Gene pulser xcell 2
Manufactured by Bio-Rad
The Gene Pulser Xcell II is a electroporation system designed for the efficient delivery of nucleic acids and other molecules into a variety of cell types. The device applies controlled electrical pulses to generate transient pores in the cell membrane, facilitating the introduction of the desired cargo into the cell.
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Lab products found in correlation
5 protocols using gene pulser xcell 2
1
CRISPR-Cas9 Mediated Knockout ESCs
2
CRISPR-Mediated Knockout of Bcl2L13 and Bnip3 in ESCs
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Bcl2L13 knockout ESCs and Bnip3 knockout ESCs were generated using the CRISPR/Cas9 system. Bcl2L13 and Bnip3 gRNA were cloned into px330 vector, and transfected into ESCs with a Gene Pulser Xcell II (Bio-Rad) according to the manufacturer’s protocols. Then knockout ESCs were identified by T7E1 enzyme and sequencing. All knockout ESCs were tested by western blotting.
3
Derivation and Characterization of Parkin and PINK1/OPTN/NDP52 Knockout ESCs
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Parkin+/+ and parkin−/− ESCs were isolated at day E3.5. The inner cell mass was picked and cultured in 2i medium. The early‐passage ESCs were cultured in 2i medium, and the later passages were maintained in ESC medium. Medium was used as described previously.14 Pink1, Optn and Ndp52 knockout ESCs were generated by CRISPR‐Cas9. We designed the gRNAs to target Pink1, Optn and Ndp52, and transfected the gRNA vector into ESCs using a Gene Pulser Xcell II (Bio‐Rad) according to the manufacturer's protocols. Knockout ESCs were identified by sequencing and western blotting.
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Parkin+/+ and parkin−/− ESCs were isolated at day E3.5. The inner cell mass was picked and cultured in 2i medium. The early‐passage ESCs were cultured in 2i medium, and the later passages were maintained in ESC medium. Medium was used as described previously.
4
Efficient Genome Editing in Fission Yeast
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All mutagenesis experiments were carried out by co-transformation of 10 µL of the pMZ374 vector series with 20 µL of the respective PCR product being used as the HR donor DNA for the target locus. Strains were grown in 5 mL of YM medium at 30 °C with shaking at 180 rpm until the culture reached an optical density (OD) at 600 nm of 0.5; cells then were transformed by electroporation using a Gene Pulser Xcell II (Bio-Rad) using standard methodologies. Transformants were spread onto EMM plates supplemented with 225 mg/L leucine and grown at 30 °C for approximately 7 days. Transformants were screened by colony PCR and DNA sequencing. For the curing of the pMZ374-based vector, the edited colony harboring the pMZ374 derivative was inoculated into YM medium, cultured for 2–3 days at 30 °C with shaking, diluted, and spread onto YM plates containing 5-fluoroorotic acid (4 mg/L). The resulting uracil auxotrophs (i.e., strains cured of the pMZ374-series plasmids) were then used in the next round of genome editing.
5
Yeast Genetic Manipulation by Co-transformation
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All mutagenesis experiments were carried out by co-transformation of 10 µL of the pMZ374 vector series with 20 μL of the respective PCR product being used as the HR donor DNA for the target locus, as described in a previous report [17 (link)]. Briefly, strains were grown in 5 mL of YM medium at 30 °C with shaking at 180 rpm until the culture reached an optical density (OD) at 600 nm of 0.5; cells then were co-transformed by electroporation using a Gene Pulser Xcell II (Bio-Rad) and standard methodologies. Transformants were selected using EMM+Leu plate, then screened by colony PCR and DNA sequencing.
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