Detecting DHFR Rearrangement
Further investigation of other unstable clones, by Southern and northern blot analyses, RT-PCR, and cloning and sequencing,
was performed to further characterize instances of DHFR rearrangement. In all cases studied (data not shown), our results indicated that the HC gene had become deleted, or "looped
out." As this phenomenon continued to be problematic, we developed the RT-PCR–based assay shown in Figure 6 to enable the
early and efficient detection of rearranged DHFR transcripts in cell line candidates.
As depicted in Figure 6A, forward and reverse primers were designed to span the HC coding sequences lost as a result of rearrangement
within our HC-DHFR-containing vector. Using these primers, RT-PCR performed on RNA prepared from clones, which had not undergone HC rearrangement,
would be expected to generate an RT-PCR product of approximately 2,700 bp. However, a rearrangement deleting any of the HC
would give rise to an RT-PCR product of less than 2,700 bp, and depending on the extent of HC sequence deletion, a product
as small as ~500 bp if only intact DHFR coding sequences remained. Using this assay, referred to as the loop out detection assay (LODA), we analyzed the RNA samples prepared from the clone described in Figure 5. As shown in Figure 6B, a ~500 bp PCR product,
indicative of HC rearrangement, was detected in the RNA sample from 63 generations. As this rearrangement was not detected
until the 100 generation time point by the northern blot in Figure 5B, LODA was more sensitive than the northern blot in detecting
this rearrangement. Additionally, LODA detected the rearrangement before the time point at which instability was marked by
a noticeable decline in Qp at ~77 generations in Figure 5A.
Figure 6. Development of the loop out detection assay (LODA). A) Schematic depicting the loop out detection assay. For a
more detailed explanation, refer to the text. B) Samples from the same RNA preparations run in the northern blot shown in
Figure 5B were subjected to the LODA depicted in Figure 6A. LODA samples were run on an agarose gel which was subsequently
stained with ethidium bromide to visualize RT-PCR products. Expected intact product of 2.7 Kb and a smaller product (~500
bp), in which the HC has been "looped out," are marked by arrows.
LODA, which is fairly high throughput, is now used at several key nodes during our cell line development platform timeline.
This reduces the probability that the development team will be investing valuable time and resources in moving forward high
producing clones that will eventually lose protein expression. In addition, LODA, coupled with sequencing, has enabled us
to catalog the sites of the HC rearrangement. This information is being used to assess whether additional modifications to
our vector strategy could result in further reductions in the frequency of HC-DHFR uncoupling.
A combination of factors
The acute instability described above is most likely the consequence of a combination of factors. One cause may be our selection
scheme, and another might be that CHO cells, although clearly a workhorse of biopharma, were not naturally designed to produce
large amounts of recombinant protein. Nonetheless, with the screening procedures and tools we have put in place, the threat
of instability to our clone selection process has been greatly reduced.