The following cell lines were donated to the DGRC by the DRSC and Perrimon lab:
S2R+-CG8786-KO (FBtc0000269)
S2R+-Tnks-KO (FBtc0000270)
These two cell lines were developed by G. Amador at the DRSC (Harvard Medical School) in collaboration with B. Xia (Perrimon lab/HMS) and Y. Ahmed (Dartmouth). The starting cell line was S2R+-MT::Cas9 (FBtc0000268). The cells were transiently transfected with a plasmid encoding an sgRNA targeting either CG8786 or Tnks and a homology donor that knocks in a selectable marker and disrupts the gene (two rounds of knockout and selection), followed by single-colony isolation and molecular characterization. Molecular evidence suggests that these isolates are knockouts of the targeted genes. The DRSC cell line IDs for these cell lines are CG8786-6 and Tnks-91. Cells are hygromycin-, puromycin- and neomycin-resistant but can be stably grown without selection or can be grown in 200 ng/uL hygromycin.
S2R+-Apc-KO-E5 (FBtc0000304)
S2R+-Apc-KO-C2
S2R+-Apc2-KO (FBtc0000267)
These three cell lines were developed by R. Viswanatha in the Perrimon lab (Harvard Medical School) in collaboration with the DRSC (HMS) and Y. Ahmed (Dartmouth). The starting cell line was S2R+-MT::Cas9 (FBtc0000268). The cells were transiently transfected with a plasmid encoding an sgRNA targeting Apc or Apc2, followed by single-colony isolation and molecular characterization. Molecular evidence suggests that these isolates are knockouts of the targeted genes. There are two distinct single-cell isolates for Apc (DRSC ID Apc-E5 and DRSC ID Apc-C2) and one for Apc2 (DRSC ID Apc2-C11).
Note: Following initial characterization of S2R+-Apc2-KO (FBtc0000267), the DRSC subsequently performed PCR amplification and next-generation sequencing of the target locus, and found evidence that some wild-type alleles remain present. This information was communicated to the DGRC and, as was appropriate based on this new information, the DGRC removed line 267 (S2R+-Apc2-KO) from the DGRC catalog.
S2R+-MT::Cas9 (FBtc0000268)
This cell line was developed by R. Viswanatha in the Perrimon lab (Harvard Medical School) to support CRISPR pooled-format screening in Drosophila cells, as described in Viswanatha et al. 2018. The starting cell line used to generate S2R+-MT::Cas9 was NPT005, which was created using MiMIC technology and is described in pubmed:22174071. N-terminally flag-tagged Cas9 driven by a metallothionein promotor was transfected into NPT005 and stable transfected cells were derived to generate S2R+-MT::Cas9. Attributes of this cell line include: S2R+ derivative; expresses mCherry ::Clic fusion protein; attP sites present; pMT-Flag-Cas9. Growth conditions: maintain hygro selection (200 ng/ mL) to prevent loss of Cas9. DRSC/Perrimon lab uses the following: Schneider’s media (Thermo 21720); 1X Penn/Strep (Thermo 15070063); 10% FBS (Thermo 16140071); 200 ng/uL (=246 uL/500 mL); Hygromycin B (Calbiochem 400051-1MU).
This work was supported in part by NIH R01 GM067761 and R24 OD019847.