CRISPR modified cell lines
We are using CRISPR gene editing technologies to generate new cell lines as part of the funded project NIH ORIP R24 OD019847 "Next-generation Drosophila cell lines to elucidate the cellular basis of human diseases" (N. Perrimon, PI; A. Simcox, Co-PI).
GFP-tagged knock-in cell lines
As part of the ORIP-funded project, we are making GFP-tagged cell lines, with an emphasis on visualization of various organelles and sub-cellular compartments. The following GFP knock-in cell lines made at the DRSC in collaboration with the Bellne lab are available for distribution by the DGRC in Bloomington, IN.
Note that the parental cell line is positive for an mCherry fusion and Cas9, as the parental cell line is S2R+-MT::Cas9 (DGRC cell catalog #268), which is described in Viswanatha et al. 2018 (PubMed ID 30051818). This parental cell line was itself derived from DRSC cell line S2R+ NPT005 (DGRC cell catalog #229), which is described in Neumuller et al. 2012 (PubMed ID 22174071).
S2R+ with GFP::Cnx99a. Ordering information: DGRC cell catalog ID #273
S2R+ with GFP::Rab11. Ordering information: DGRC cell catalog ID #274
S2R+ with GFP::Polo. Ordering information: DGRC cell catalog ID #275
S2R+ with GFP::Gmap (clone #4). Ordering information: DGRC cell catalog ID #276
S2R+ with GFP::Gmap (clone $7). Ordering information: DGRC cell catalog ID #277
S2R+ with GFP::Fib (clone #11). Ordering information: DGRC cell catalog ID #278
S2R+ with GFP::Fib (clone #12). Ordering information: DGRC cell catalog ID #279
S2R+ with GFP::Golgin. Ordering information: DGRC cell catalog ID #280
S2R+ with GFP::Arl8. Ordering information: DGRC cell catalog ID #291
S2R+ with GFP::Lam. Ordering information: DGRC cell catalog ID #292
S2R+ with GFP::Spin. Ordering information: DGRC cell catalog ID #293
S2R+ with GFP::Sec23. Ordering information: DGRC cell catalog ID #294
S2R+ with GFP::Tom20. Ordering information: DGRC cell catalog ID #302
These cell lines were made using constructs designed and provided by Kanca and Bellen (Baylor College of Medicine). The cell lines were engineered, isolated, and validated at the DRSC. Validation testing included live-cell imaging, fixed-cell imaging (co-stained with an antibody, when possible), and molecular characterization of the insertion endpoints.
See the file attached below for a slide presentation with images of live and fixed cells, as well as other relevant information about the approach and the resulting GFP-tagged cell lines.
In addition, C-terminal GFP knock-in cell lines were generated as described in a BioRxiv preprint from Bosch et al. (2019) using an 'armless' donor approach. Act5c::GFP, Tub84B::GFP, His2Av::GFP, and Lamin::GFP fusion cell lines were made using this approach and are being shared with the DGRC for distribution to the community.
Knockout cell lines
S2R+-ZnT63C-KO, NHEJ-mediated knockout of ZnT63C. As described in PMID: 29223976. Ordering information: DGRC cell catalog #265.
S2R+-IA2-KO, NHEJ-mediated knockout of ia2. As described in PMID: 29223976. Ordering information: DGRC cell catalog #266.
S2R+-Apc-KO, two independent cell lines made with one method, DGRC cell catalog #271 and DGRC cell catalog #272
S2R+-Apc-KO, one additional cell line made with a different method, DGRC cell catalog #270
S2R+-gig-KO, DGRC cell catalog #297
S2R+-hairy-KO, DGRC cell catalog #298
S2R+-Moe-KO, DGRC cell catalog #303
S2R+-Pex19-KO, DGRC cell catalog #306
S2R+-Pten-KO, DGRC cell catalog #307
S2R+-Slik-KO, DGRC cell catalog #308
S2R+-Tnks-KO, three independent cell lines, DGRC cell catalog #299, DGRC cell catalog #300, and DGRC cell catalog #301
S2R+-Tnks-KO, one additional cell line made with a different method, DGRC cell catalog #304
S2R+-TSC1-KO, DGRC cell catalog #305
S2R+-Yki-KO, DGRC cell catalog #309
These cell lines have been sequence verified as containing only knockout alleles by PCR amplification of the target region followed by next-generation sequencing of the PCR amplicon, contig assembly, and comparison with the wild-type reference sequence (or, for KO alleles generated via knock-in, verified using PCR validation of the insertion allele). We wanted to make sure that the distribution copies of the cell lines are correct. To do this, for a subset of these cell lines, (1) the DGRC prepared genomic DNA from their distribution copies of the cell lines, (2) they shipped that gDNA to the DRSC/TRiP, and (3) we used that gDNA as template for PCR and NGS, and validated that all alleles are predicted to be gene knockout alleles. Information relevant to re-validation in your own lab is provided at the DGRC website.
Did you request GFP-tagged or KO cells from the DGRC and use them in a study? If so, please acknowledge both the cell line developers (DRSC) and the distribtors (DGRC) by citing NIH Grant 5R24OD019847, which supported production of the resource at DRSC/TRiP, and the Drosophila Genome Resource Center, NIH grant 2P40OD010949, as well as relevant pulications.
Slide presentation file -- GFP-tagged cell lines:
smohr_drsc_gfp-tag_websharecopy.pdf | 12.23 MB |