In our experience a significant number of Saccharomyces cerevisiae knock-out strains in the BY4741 (MAT a) and BY4742 (MAT alpha) genetic background still carry a copy of the wild-type gene. We test all strains by PCR using primers flanking and inside the ORF of interest and the KAN cassette. Several supposedly KO strains are either wild-type or contain both the WT and KAN ORF. The strains are currently distributed in United States by Horizon, previously by Dharmacon and Open Biosystem. Although the catalog number varies, the clone ID remains the same. In Europe the strains are distributed by EUROSCARF (although we have not directly tested strains obtained from EUROSCARF).
We are proposing to establish a resource for the yeast community where researchers can indicate which strains (from any collection) they have tested and found to be incorrect.
Below is the list of the strains tested in our lab that retain the wild-type gene.
Please take a moment to post the information regarding S. cerevisiae strains from available knock-out collections which you have tested and found incorrect.
- BY4741 KO aco1: ORF YLR304C. Horizon cat# YSC6273-201936681. Clone ID 5212
- BY4741 KO afg3: ORF YER017C. Horizon cat# YSC6273-201920860. Clone ID 148
- BY4741 KO aim26: ORF YKL037W. Horizon cat# YSC6273-201930575. Clone ID 3104
- BY4741 KO atg8: ORF YBL078C. Horizon cat# YSC6273-201936426. Clone ID 4886
- BY4741 KO emi1: ORF YDR512C. Horizon cat# YSC6273-213589048. Clone ID 34346
- BY4741 KO erg6: ORF YML008C. Horizon cat# YSC6273-201937025. Clone ID 568
- BY4741 KO cor1: ORF YBL045C. Horizon cat# YSC6273-201930334. Clone ID 3071
- BY4741 KO cox17: ORF YLL009C. Horizon cat# YSC6273-201920991. Clone ID 1497
- BY4742 KO cox17: ORF YLL009C. Horizon cat# YSC6272-201918401. Clone ID 11497
- BY4741 KO cyt1: ORF YOR065W. Horizon cat# YSC6273-201923155. Clone ID 1841
- BY4742 KO mam33: ORF YIL070C. Horizon cat# YSC6272-201918373. Clone ID 11463
- BY4741 KO mrp17: ORF YKL003C. Horizon cat# YSC6273-201936404. Clone ID 4851
- BY4741 KO mrps5: ORF YBR251W. Horizon cat# YSC6273-201932660. Clone ID 3391
- BY4741 KO mrpl24: ORF YMR193W. Horizon cat# YSC6273-201938490. Clone ID 778
- BY4741 KO mrpl31: ORF YKL138C. Horizon cat# YSC6273-201936510. Clone ID 4988
- BY4741 KO mrpl36: ORF YBR122C. Horizon cat# YSC6273-201931704. Clone ID 3261
- BY4741 KO mrpl49: ORF YJL096W. Horizon cat# YSC6273-201919637. Clone ID 1327
- BY4741 KO pet191: ORF YJR034W. Horizon cat# YSC6273-213589148. Clone ID 36832
- BY4742 KO qcr2: ORF YPR191W. Horizon cat# YSC6272-201921493. Clone ID 15606
- BY4742 KO qcr8: ORF YJL166W. Horizon cat# YSC6272-201918218. Clone ID 11259
- BY4741 KO rip1: ORF YEL024W. Horizon cat# YSC6273-201928856. Clone ID 265
- BY4742 KO rmd9: ORF YGL107C. Horizon cat# YSC6272-201920604. Clone ID 14474
- BY4741 KO rvs161: ORF YCR009C. Horizon cat# YSC6273-201933442. Clone ID 3489
2023-11-20T05:00:00Z
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Totally agree, I think that such a resource would be extremely useful. I would suggest to cross-reference individual reports of strain issues to the Saccharomyces Genome Variation database (https://www.sgv.gurdon.cam.ac.uk), which hosts whole genome sequences of the BY4743 homozygous knock-out strains, and our Yeast Phenome database (https://www.yeastphenome.org), which reports all published phenotypes for BY4741, BY4742 and BY4743 strains.
Hallo, for the MATa collection, the proteome (doi: 10.1016/j.cell.2023.03.026) provides such a validation, at least for the knock-outs that encode for proteins that are concentrated high enough to be reliably detected in the high throughput proteomic approach we used (about ~50% of expressed genes, these account for ~80%-90% of the total proteomic mass.). The complete dataset can be downloaded here 5k_dataset.complete.csv - Google Drive . Albeit the proteome is not covering all genes, it has the advantage over PCR testing that it is not sidelined by copy number varations and aneuploidies, which are one posssible cause of why a correclty deleted gene might still be present.
Thank you very much for sharing the information and very valuable resource. You reported in the manuscript that you were able to validate 960 strains from the KO collection and in 839 the delated protein was not detected. It would be great, if you could share a list of the 121 putative KO strains in which you detected the deleted protein at wild-type or reduced level, so that the information is easily accessible. I think that it would be really beneficial, especially for researchers working on individual pathways, if we as a community were able to compile a complete list of strains from the KO collection where the putative delete gene and/or protein is still present at any level. This would save time and funds and reduce the risk of mayor errors. Thank you again very much for your contribution.
Please have a look on this table (link below). When a supposedly deleted gene is still detected (‘Not missing’), the table distinguishes if the protein is expressed at wild type levels (‘not DE’), in which case the strain ist most likely wrong and looks like wild-type, - or whether it is detected, but at a much different level as in the wild type (‘DE’). If the latter, one is most likely dealing with a copy number variaion, translocation, aneuploidy, or perhaps cross-contamination.
https://data.mendeley.com/datasets/w8jtmnszd9/2/files/6a6cc1ec-35cc-4f52-864b-8f07433453ed
This is very useful. Thank you very much.
Here are a few more KO strains in which a copy of the wild-type gene is still present, validated by Khalimonchuk lab:
BY4741 KO pet191: ORF YJR034W. Clone ID 36832
BY4741 KO dpc29 (alias Hah1): ORF YGR021W. Clone ID 4651
BY4741 KO mpa43: ORF YNL249C. Clone ID 1195
BY4741 KO fmp40: ORF YPL222W. Clone ID 1070