The HEK293 human cell lineage is widely used in cell biology
The HEK293 human cell lineage is widely used in cell biology and biotechnology. from your kidney of an aborted human being embryo of unknown parenthood by transformation with sheared Adenovirus 5 DNA. The human being embryonic kidney cells at 6-Maleimidocaproic acid manufacture first seemed recalcitrant to transformation. 6-Maleimidocaproic acid manufacture After many efforts, cell growth took off only several months after the isolation of a single transformed clone. This cell collection is known as HEK293 or 293 cells (ATCC accession quantity CRL-1573). A 4-kbp adenoviral genome fragment is known to possess integrated in chromosome 19 (ref. 4) and encodes for the E1A/E1B proteins, which interfere with the cell cycle control pathways and counteract apoptosis5,6. Cytogenetic analysis established the 293 collection is pseudotriploid7. Given the broad use of 293 cells for biomedical study and computer virus/protein production, we decided to perform a comprehensive genomic characterization of the 293 cell collection and the most commonly used derived lines (Fig. 1a) to better understand the dynamics of the 293 genome under the methods commonly used in biotechnological executive of mammalian cell lines. Number 1 HEK293 cell collection manifestation profiling. First among these derived lines, we analysed 293T, which expresses a temperature-sensitive allele of the SV40 T antigen8,9. This enables the amplification of vectors comprising the SV40 ori and thus considerably increases the manifestation levels acquired with transient transfection. SV40 T forms a complex with and inhibits p53, probably further diminishing genome integrity10. The original 293 collection was suspension growth-adapted through serial passaging in Jokliks altered minimal Eagles medium11. Full adaptation required about 7 weeks, and the 1st passages were so difficult the few cells that grew through are likely to have been almost clonal (Dr Bruce Stillman, personal communication). The fully adapted 6-Maleimidocaproic acid manufacture cell collection is known as 293S and is also analysed here. Subsequently, this collection was mutagenized with ethylmethanesulfonate (EMS) and a Ricin toxin-resistant clone was selected out. The collection lacked N-acetylglucosaminyltransferase I activity (encoded from the gene) and accordingly mainly modifies glycoproteins with the Man5GlcNAc2 N-glycan. Then, a stable tetR repressorCexpressing clone of this glyco-engineered Rabbit Polyclonal to SIRPB1 cell collection was derived to enable tetracyclin-inducible protein manifestation12. This cell collection is definitely widely used for the production of homogenously N-glycosylated proteins and will be referred to as 293SG. Apart from these four cell lines in common use, we also analysed the genome of two 293-derived lines used in our laboratory for proteinCprotein connection testing (293FTM) and glyco-engineering (293SGGD; details in Supplementary Info). In our study, following genomic studies of other human being cell lines13,14,15, we aim to provide a full-genome source for these cell biology workhorse cell lines while developing the necessary tools to make such resources easily available. This enables all experts using the 293 cell lines to make fully educated analyses of genomic regions of interest to their studies, without expert bioinformatics skills. We also map the genomic changes accumulating after standard laboratory cell culturing (passaging and freezing), providing a way to assess genomic stability of each collection. Furthermore, we present a workflow for determining the insertion sites of viral sequences and plasmids based on the genome sequencing data. The intense chromosome structure diversity/plasticity in the 293 cell collection underlies a novel application: selection of 293 clones surviving stringent selective conditions (in our case: ricin toxin), followed by whole-genome analysis of copy quantity alterations, can efficiently pinpoint the genomic region(s) that contain the gene(s) that is required for adaptation to the people selective conditions. Results 293 cell lineage genome, karyotype and transcriptome For genome resequencing, we used total genomics (CG) high-coverage genome sequencing technology16 (Supplementary Methods; data set summary in Supplementary Furniture 1 and 2, and sequencing quality overview in Supplementary Fig. 1). 293 cells are of female provenance, once we find no trace of Y-chromosome-derived sequence in our data models. The mitochondrial sequence belongs to the oldest Western haplogroup U5a1 (refs 17, 18). Furthermore, we applied multiplex fluorescence hybridization analysis to our 293 lines (Supplementary Data 1). A wide diversity of karyotypes was found, also within each clone, with some chromosomal alterations relative to the human research genome present in almost all.