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P3 primary buffer

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P3 primary buffer is a laboratory solution used in various bioprocessing applications. It serves as a fundamental component in maintaining the appropriate pH and ionic environment for biological samples and processes.

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Lab products found in correlation

4d nucleofector, by Lonza (9 mentions) Versene, by Thermo Fisher Scientific (8 mentions) Mtesr plus, by STEMCELL (8 mentions) Geneart, by Thermo Fisher Scientific (1 mentions) Cytotune ips 2.0 sendai reprogramming kit, by Thermo Fisher Scientific (1 mentions)

9 protocols using p3 primary buffer

1

Generating iPSC Clones with FUS P525L Mutation

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As previously described (Akter et al., 2023 (link)), human FUS P525L mutant clones were generated from the healthy iPSC line by Genome Engineering and iPSC Center (GEiC) at Washington University in St. Louis. To begin, about 1 million single cells were suspended in P3 primary buffer (Lonza), along with a gRNA/Cas9 ribonuclease protein (RNP) complex consisting of 200 pmol synthetic gRNA, 80 pmol SpCas9 protein, and hFUS mutant ssODN (Table 2). The cells were then electroporated with a 4DNucleofector (Lonza) using the CA-137 program. The editing efficiency was verified after nucleofection by targeted deep sequencing, using specific primer sets for the target regions, and the pool was sorted by single cells. Targeted deep sequencing analysis was employed to screen the single-cell clones. All iPSCs were cultured using mTeSR Plus (STEMCELL Technology) on Matrigel-coated plates at 37 °C in a humidified, 5% CO2 incubator. Passage was performed at a 1:6 ratio using a gentle cell dissociation reagent (Versene, Gibco).
Akter M., Cui H., Hosain M.A, & Ding B. (2023). Generation of two induced pluripotent stem cell lines with heterozygous and homozygous amyotrophic lateral sclerosis-causing mutation P525L (c.1574C > T) in FUS gene. Stem cell research, 69, 103103.
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2

Generating iPSC Clones with FUS P525L Mutation

Cited 1 time
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As previously described (Akter et al., 2023 (link)), human FUS P525L mutant clones were generated from the healthy iPSC line by Genome Engineering and iPSC Center (GEiC) at Washington University in St. Louis. To begin, about 1 million single cells were suspended in P3 primary buffer (Lonza), along with a gRNA/Cas9 ribonuclease protein (RNP) complex consisting of 200 pmol synthetic gRNA, 80 pmol SpCas9 protein, and hFUS mutant ssODN (Table 2). The cells were then electroporated with a 4DNucleofector (Lonza) using the CA-137 program. The editing efficiency was verified after nucleofection by targeted deep sequencing, using specific primer sets for the target regions, and the pool was sorted by single cells. Targeted deep sequencing analysis was employed to screen the single-cell clones. All iPSCs were cultured using mTeSR Plus (STEMCELL Technology) on Matrigel-coated plates at 37 °C in a humidified, 5% CO2 incubator. Passage was performed at a 1:6 ratio using a gentle cell dissociation reagent (Versene, Gibco).
Akter M., Cui H., Hosain M.A, & Ding B. (2023). Generation of two induced pluripotent stem cell lines with heterozygous and homozygous amyotrophic lateral sclerosis-causing mutation P525L (c.1574C > T) in FUS gene. Stem cell research, 69, 103103.
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3

Generation of WNT9B-mCerulean3 Tagged iPSCs

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BJFF.6 iPSCs was generated from human BJ (ATCC ID: CRL-2522) by the Genome Engineering and iPSC Center (GEiC) at Washington University (St. Louis) using the CytoTune-iPS 2.0 Sendai reprogramming kit (ThermoFisher) following the manufacturer’s recommended protocol. This line is described on the RBK website (https://www.rebuildingakidney.org/chaise/record/#2/Cell_Line:Parental_Cell_Line/RID=Q-2D6W). The WNT9B C-terminal mCerulean3 tagged cell line (WNT9BmCerulean) was generated by GEiC using the BJFF.6 iPSC line. Approximately 1 to 1.5 × 106 hPSCs were washed in DPBS and resuspended in P3 primary buffer (Lonza) with 1 μg of a WNT9B-specific gRNA (5’-AGACTGGCTTGCTGGGCAGT-3’) expression plasmid (cloned into MLM3636), 1.5 μg of the Cas9 expression vector, p3s-Cas9HC, and 1.5 ug of hWNT9B-mCerulean3 donor plasmid (GeneArt, ThermoFisher). The cells were subsequently electroporated with a 4D-Nucleofector (Lonza) using the CA-137 program. Following nucleofection, cells were then single-cell sorted as previously described (Chen and Pruett-Miller, 2018 (link)). Single cell clones were expanded and screened by PCR using primer sets that flank the 5’ (WNT9B_5’F and mCeruleanR) and 3’ (mCeruleanF and WNT9B_3’R) recombination junctions. The WNT9B-mCerulean3 tagged region of successfully targeted clones was also verified by Sanger sequencing.
Howden S.E., Wilson S.B., Groenewegen E., Starks L., Forbes T.A., Tan K.S., Vanslambrouck J.M., Holloway E.M., Chen Y.H., Jain S., Spence J.R, & Little M.H. (2020). Plasticity of distal nephron epithelia from human kidney organoids enables the induction of ureteric tip and stalk.. Cell stem cell, 28(4), 671-684.e6.
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4

Generation of FUS R521G hiPSC Clones

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Human FUS R521G mutant clones were generated from a healthy hiPSC line by Genome Engineering and iPSC Center (GEiC) at Washington University in St. Louis. Briefly, approximately 1 × 106 single cells were resuspended in P3 primary buffer (Lonza) with gRNA/Cas9 ribo-nuclease protein (RNP) complex (200 pmol synthetic gRNA and 80 pmol SpCas9 protein) and hFUS mutant ssODN (Table 2). Subsequently, cells were electroporated with a 4DNucleofector (Lonza) using CA-137 program. Following nucleofection, the editing efficiency was confirmed by targeted deep sequencing using primer sets specific to target regions and then the pool was single-cell sorted. Single-cell clones were screened with targeted deep sequencing analysis. All iPSCs were cultured with mTeSR Plus (STEMCELL Technology) on Matrigel-coated plates at 37 °C in a humidified, 5% CO2 incubator and passage at a 1:6 ratio using gentle cell dissociation reagent (Versene, Gibco).
Boeuf H., Reimund B., Jansen-Durr P, & Kédinger C. (1990). Differential activation of the E2F transcription factor by the adenovirus EIa and EIV products in F9 cells.. Proceedings of the National Academy of Sciences of the United States of America, 87(5), 1782-1786.
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5

Generation of FUS R521G hiPSC Clones

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Human FUS R521G mutant clones were generated from a healthy hiPSC line by Genome Engineering and iPSC Center (GEiC) at Washington University in St. Louis. Briefly, approximately 1 × 106 single cells were resuspended in P3 primary buffer (Lonza) with gRNA/Cas9 ribo-nuclease protein (RNP) complex (200 pmol synthetic gRNA and 80 pmol SpCas9 protein) and hFUS mutant ssODN (Table 2). Subsequently, cells were electroporated with a 4DNucleofector (Lonza) using CA-137 program. Following nucleofection, the editing efficiency was confirmed by targeted deep sequencing using primer sets specific to target regions and then the pool was single-cell sorted. Single-cell clones were screened with targeted deep sequencing analysis. All iPSCs were cultured with mTeSR Plus (STEMCELL Technology) on Matrigel-coated plates at 37 °C in a humidified, 5% CO2 incubator and passage at a 1:6 ratio using gentle cell dissociation reagent (Versene, Gibco).
Akter M., Cui H., Hosain A, & Ding B. (2023). Generation of two induced pluripotent stem cell lines with heterozygous and homozygous amyotrophic lateral sclerosis-causing mutation R521G (c.1561C > G) in FUS gene. Stem cell research, 69, 103078.
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6

CRISPR-Mediated Correction of TOR1A Mutation

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The GAG mutation in the TOR1A gene in DYT1 hiPSC was genetically corrected at the Genome Engineering and iPSC Center (GEiC) at Washington University in St. Louis. Briefly, approximately 1 × 106 single cells were resuspended in P3 primary buffer (Lonza) with gRNA/Cas9 ribonuclease protein (RNP) complex (200 pmol synthetic gRNA and 80 pmol HiFi Cas9 protein) and TOR1A correction ssODN (Table 2). A silent mutation (C to T) was introduced in the donor oligo sequence (ssODN) to avoid the re-cutting of the edited sequence by CRISPR/Cas9. Subsequently, cells were electroporated with a 4DNucleofector (Lonza) using the CA-137 program. Following nucleofection, the editing efficiency was confirmed by targeted deep sequencing using primer sets specific to target regions and then the pool was single-cell sorted. Single cell clones were screened with targeted deep sequencing analysis. All iPSCs were cultured with mTeSR Plus (STEMCELL Technology) on Matrigel-coated plates at 37 °C in a humidified, 5% CO2 incubator and passage at a 1:6 ratio using gentle cell dissociation reagent (Versene, Gibco).
Akter M., Cui H., Chen Y.H, & Ding B. (2022). Generation of gene-corrected isogenic control cell lines from a DYT1 dystonia patient iPSC line carrying a heterozygous GAG mutation in TOR1A gene. Stem cell research, 62, 102807.
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7

CRISPR-Mediated Gene Editing of iPSCs

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Human TOR1A mutant clones were generated from the WTC11 iPSC line (Gladstone institute, UCSFi001-A) by the Genome Engineering and iPSC center (GEiC) at Washington University in St. Louis. Briefly, approximately 1×106 single cells were resuspended in P3 primary buffer (Lonza) with gRNA/Cas9 ribonucleoprotein (RNP) complex (200 pmol synthetic gRNA and 80 pmol SpCas9 protein) and hTOR1A mutant single-stranded oligo donor (ssODN) (Table 2). Subsequently, cells were electroporated with a 4D-Nucleofector (Lonza) using CA-137 program. Following nucleofection, the editing efficiency was confirmed by targeted deep sequencing using primer sets specific to target regions, and then the pool was single-cell sorted. Single cell clones were screened with targeted deep sequencing analysis. All iPSCs were cultured with mTeSR Plus (STEMCELL Technology) on matrigel-coated plates at 37 °C in a humidified, 5% CO2 incubator and passage at a 1:6 ratio using gentle cell dissociation reagent (Versene, Gibco).
Akter M., Cui H., Chen Y.H, & Ding B. (2021). Generation of two induced pluripotent stem cell lines with heterozygous and homozygous GAG deletion in TOR1A gene from a healthy hiPSC line. Stem cell research, 56, 102536.
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8

CRISPR-Mediated Gene Editing of iPSCs

Check if the same lab product or an alternative is used in the 5 most similar protocols
Human TOR1A mutant clones were generated from the WTC11 iPSC line (Gladstone institute, UCSFi001-A) by the Genome Engineering and iPSC center (GEiC) at Washington University in St. Louis. Briefly, approximately 1×106 single cells were resuspended in P3 primary buffer (Lonza) with gRNA/Cas9 ribonucleoprotein (RNP) complex (200 pmol synthetic gRNA and 80 pmol SpCas9 protein) and hTOR1A mutant single-stranded oligo donor (ssODN) (Table 2). Subsequently, cells were electroporated with a 4D-Nucleofector (Lonza) using CA-137 program. Following nucleofection, the editing efficiency was confirmed by targeted deep sequencing using primer sets specific to target regions, and then the pool was single-cell sorted. Single cell clones were screened with targeted deep sequencing analysis. All iPSCs were cultured with mTeSR Plus (STEMCELL Technology) on matrigel-coated plates at 37 °C in a humidified, 5% CO2 incubator and passage at a 1:6 ratio using gentle cell dissociation reagent (Versene, Gibco).
Akter M., Cui H., Chen Y.H, & Ding B. (2021). Generation of two induced pluripotent stem cell lines with heterozygous and homozygous GAG deletion in TOR1A gene from a healthy hiPSC line. Stem cell research, 56, 102536.
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9

CRISPR-Mediated Correction of TOR1A Mutation

Check if the same lab product or an alternative is used in the 5 most similar protocols
The GAG mutation in the TOR1A gene in DYT1 hiPSC was genetically corrected at the Genome Engineering and iPSC Center (GEiC) at Washington University in St. Louis. Briefly, approximately 1 × 106 single cells were resuspended in P3 primary buffer (Lonza) with gRNA/Cas9 ribonuclease protein (RNP) complex (200 pmol synthetic gRNA and 80 pmol HiFi Cas9 protein) and TOR1A correction ssODN (Table 2). A silent mutation (C to T) was introduced in the donor oligo sequence (ssODN) to avoid the re-cutting of the edited sequence by CRISPR/Cas9. Subsequently, cells were electroporated with a 4DNucleofector (Lonza) using the CA-137 program. Following nucleofection, the editing efficiency was confirmed by targeted deep sequencing using primer sets specific to target regions and then the pool was single-cell sorted. Single cell clones were screened with targeted deep sequencing analysis. All iPSCs were cultured with mTeSR Plus (STEMCELL Technology) on Matrigel-coated plates at 37 °C in a humidified, 5% CO2 incubator and passage at a 1:6 ratio using gentle cell dissociation reagent (Versene, Gibco).
Akter M., Cui H., Chen Y.H, & Ding B. (2022). Generation of gene-corrected isogenic control cell lines from a DYT1 dystonia patient iPSC line carrying a heterozygous GAG mutation in TOR1A gene. Stem cell research, 62, 102807.
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