Table 1.
Oligonucleotides and probes.
Fig 1.
Configuration of immunoglobulin heavy chain (HC) and light chain (LC) antibody sequences.
Diagrams illustrating the configuration of the immunoglobulin heavy chain (HC) and light chain (LC) antibody gene sequences relative to the location of the chromatin function modifying elements inserted within the vector. Top line: control plasmid vector showing in order hCMV promoter (green arrow)-LC gene cassette (tandem brown and hashed arrows), hCMV-HC gene unit. Sites of insertion of the chromatin function modifying elements are denoted as blue arrows.
Fig 2.
A2UCOE-augmented vectors provide the highest elevation in antibody production in stably transfected cell pools.
The control and chromatin element containing vectors as depicted in Fig 1, were introduced into CHO-K1 cells and stably transfected cell pools selected with G418. Cells were then seeded at 3x105 cells/ml and antibody production measured after 10 days using a mouse IgG ELISA. Each data point represents mean +/- SD (n = 2). (A) 1.5kb A2UCOE, (B) MAR X_S29, (C) STAR40, (D) cHS4.
Fig 3.
A2UCOE provides higher mean antibody expression levels in stably transfected CHO-K1 cells clones.
From a total of 192 initially selected clones harbouring either expression vectors containing the control or chromatin elements 24, clones with the highest expression levels from each set were seeded into 24 well tissue culture plates and antibody production measured 10 days later. Graphs show the range of expression levels with the mean expression level highlighted by horizontal bar for each cell line for the four elements. A multi-comparison 1-way ANOVA Dunnetts analysis was performed with 95% CI * p<0.05, ** p<0.01 and *** p<0.001. (A) 1.5kb A2UCOE, (B) MAR X_S29, (C) cHS4, (D) STAR 40.
Fig 4.
A2UCOE-augmented vectors provide higher antibody expression in CHO-K1 cells following GS selection.
The A2UCOE (3’ HC 5’ LC 5’ HC), MAR X_S29 (3’ HC 5’LC) and cHS4 (5’LC 5’HC) constructs that gave the highest yields following G418 selection (Fig 2) were stably transfected and selected using the GS system. Cells were then seeded at 3x105 cells/ml and expression measured after 10 days using a mouse IgG ELISA. Each data point represents mean +/- SD (n = 3) of three independent transfections.
Fig 5.
Comparative analysis of antibody yields from clones selected using the GS selection system.
Following an initial isolation of 192 clones from the stably transfected CHO-K1GS selected pools containing either the control or 1.5kb A2UCOE (3’ HC 5’ LC 5’ HC), MAR X_S29 (3’ HC 5’LC) and cHS4 (5’LC 5’HC) constructs (Fig 4), the 48 highest expressing clones were seeded and expanded in 24 well tissue culture plates and grown for 10 days. Supernatant was then assayed for antibody production using a mouse IgG ELISA. As can be seen clones containing the 1.5kb A2UCOE construct provide a highest overall level of antibody production. The percentage distribution of clones in each quartile is shown for each element.
Fig 6.
Southern blot analysis of control and A2UCOE vector-containing clones.
Total genomic DNA from CHO-K1 clones harbouring stably transfected 1.5kb A2UCOE (3’ HC 5’ LC 5’ HC) and control vectors were digested with EcoRI restriction enzyme and subjected to Southern blot analysis with hybridisation using a DIG labelled probe spanning the ampicillin resistance gene on the plasmid vector backbone. (A) C1–C10 = control vector clones. (B) U1–U10 = A2UCOE (3’ HC 5’ LC 5’ HC) vector clones. M = DIG labelled DNA size (kb) ladder. K1 = untransfected CHO-K1 gDNA. 108 = 1 x 108 plasmid copies. 106 = 1 x 106 plasmid copies.
Table 2.
Summary of cell line families harbouring the HC and LC antibody control (C) and A2UCOE (U) vectors as determined by Southern blot Analysis.
Fig 7.
Chromosomal integration sites by Fluorescence In Situ Hybridisation.
Biotinylated probes were generated using the 1.5kb A2UCOE plasmid and hybridised to metaphase spreads for the three 1.5kb A2UCOE (3’ HC 5’ LC 5’ HC) construct-containing CHO-K1 clones (Fig 6). Sites of probe hybridisation were detected with streptavidin conjugated to Cy3 and integration sites are revealed as pink dots. Chromosomes were highlighted by counterstaining with DAPI. Panel A: clone U2; Panel B: clone U8; Panel C: clone U9. Panel D: untransfected CHO-K1 cells.
Fig 8.
Stability of antibody expression from clones harbouring control and A2UCOE (3’ HC 5’ LC 5’ HC) vectors cultured in the absence of drug selective pressure.
Stably transfected CHO-K1 clones containing the control and 1.5kb A2UCOE (3’ HC 5’ LC 5’ HC) vectors (Fig 6) were continuously cultured for 120 generations without MSX selective pressure. Overgrow cultures of 10 day duration were set up at 0, 40, 80 and 120 generations. Supernatant was then assayed by mFc ELISA to establish antibody expression levels. The percentage of expression remaining after 120 generations is also shown. (A) control vector (B) 1.5kb A2UCOE construct. Each data point represents mean +/- SD of triplicate determinations.
Fig 9.
Relative mRNA levels parallel antibody production during long term culture in the absence of selection pressure.
Relative HC and LC mRNA levels were quantified at 0, 40, 80 and 120 generations using RT-Q-PCR. RQ values were calculated using the 2-ΔΔ CT method. Input RNA quantity was normalised using GAPDH as an internal control. Each data point represents mean +/- SD of two independent RT-QPCR assays.
Fig 10.
DNA methylation status of CpG dinucleotides within the CMV promoter regions driving HC and LC expression in (A) control (B) A2UCOE (3’ HC 5’ LC 5’ HC) clones.
Bisulphite conversion and DNA sequencing was performed to establish the number of methylated cytosine residues in CpG dinucleotides within the hCMV-MIE promoters. Following bisulphite conversion, the hCMV-MIE regions were amplified by PCR and products cloned and sequenced. Each line represents the sequence of an individual PCR amplified plasmid clone from the hCMV-MIE region. Each square on a given line represents the position of a CpG dinucleotide within the amplified hCMV-MIE region. White squares, unmethylated CpG dinucleotide; black squares, methylated CpG dinucleotide.