Lrg2.1 mcherry vector

Manufactured by Addgene

The LRG2.1 mCherry vector is a plasmid used for the expression of the mCherry fluorescent protein in cells. The mCherry protein is a red fluorescent protein derived from the DsRed protein of the sea anemone Discosoma. This vector can be used for various applications, such as tracking protein localization or monitoring gene expression in live cells.

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Lab products found in correlation

Stbl3 e coli, by Thermo Fisher Scientific (1 mentions) Dcas9 krab construct, by Addgene (1 mentions)

2 protocols using lrg2.1 mcherry vector

CRISPRi Guide RNA Cloning and Validation

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Guide RNAs for CRISPRi experiments were chosen from refs^{60 (link),97}, and two guides per gene were cloned into the LRG2.1 mCherry vector (Addgene #108099) as described above. Plasmids were transformed in Stbl3 E. coli and sequenced to confirm the presence of the correct gRNA sequence. CRISPRi gRNA sequences are listed in Table S5B.

Girish V., Lakhani A.A., Scaduto C.M., Thompson S.L., Brown L.M., Hagenson R.A., Sausville E.L., Mendelson B.E., Lukow D.A., Yuan M.L., Kandikuppa P.K., Stevens E.C., Lee S.N., Salovska B., Li W., Smith J.C., Taylor A.M., Martienssen R.A., Liu Y., Sun R, & Sheltzer J.M. (2023). Oncogene-like addiction to aneuploidy in human cancers. bioRxiv.

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CRISPRi Guide RNA Cloning and Lentivirus Production

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Guide RNAs for CRISPRi experiments were chosen from (Horlbeck et al. 2016 (link)). Guides were cloned into the LRG2.1 mCherry vector (Addgene; Cat. No. 108099) using a BsmBI digestion as previously described (Giuliano et al. 2019 ). Plasmids were transformed in Stbl3 E. coli (Thermo Fisher; Cat. No. C737303) and sequenced to confirm the presence of the correct gRNA sequence. A dCas9-KRAB construct (Addgene; Cat. No. 85969) was used to knock down target gene expression. Guide RNA sequences are listed in Table S1B.
Lentivirus was generated using calcium phosphate transfection as previously described (Chang et al. 2013 (link)). Supernatant was harvested at 2 to 3 intervals 48 to 72 hr post-transfection by filtering through a 0.45 μm syringe, and then frozen at −80° C for later use or supplied directly to cells with 4 μg/mL polybrene.

Vasudevan A., Baruah P.S., Smith J.C., Wang Z., Sayles N.M., Andrews P., Kendall J., Leu J., Chunduri N.K., Levy D., Wigler M., Storchová Z, & Sheltzer J.M. (2020). Single chromosomal gains can function as metastasis suppressors and promoters in colon cancer. Developmental cell, 52(4), 413-428.e6.

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