Combining high-throughput screening of caspase activity with anti-apoptosis genes for development of robust CHO production cell lines.

A set of anti-apoptotic genes were over-expressed, either singly or in combination, in an effort to develop robust Chinese Hamster Ovary host cell lines suitable for manufacturing biotherapeutics. High-throughput screening of caspase 3/7 activity enabled a rapid selection of transfectants with reduced caspase activity relative to the host cell line. Transfectants with reduced caspase 3/7 activity were then tested for improved integrated viable cell count (IVCC), a function of peak viable cell density and longevity. The maximal level of improvement in IVCC could be achieved by over-expression of either single anti-apoptotic genes, e.g., Bcl-2Δ (a mutated variant of Bcl-2) or Bcl-XL, or a combination of two or three anti-apoptotic genes, e.g., E1B-19K, Aven, and XIAPΔ. These cell lines yielded higher transient antibody production and a greater number of stable clones with high antibody yields. In a 5 L fed-batch bioreactor system, BΔ31-1, a stable clone expressing Bcl-2Δ, had a product titer that was 180% as compared to an optimal clone (Con-1) from the control cell line. Although lactate accumulated to more than 5 g/L in the control culture, its concentration was reduced in the anti-apoptotic BΔ31-1 cultures to below 1 g/L, confirming our earlier findings that cells over-expressing anti-apoptotic genes consume the lactate that would otherwise accumulate as a by-product in the culture medium. To the best of our knowledge, this is the first study to use the high throughput caspase screening method to identify CHO host cell lines with superior anti-apoptotic characteristics.

Regulation of photoreceptor Per1 and Per2 by light, dopamine and a circadian clock.

In the Xenopus laevis retina, a principal model for retinal circadian organization, photoreceptors have all the properties of circadian oscillators. However, rhythmic oscillations of Per1 gene expression in the inner retina (but not photoreceptors) have been reported in mice with the suggestion that mice and frogs have a different retinal circadian organization. Although it is known that two period genes (xPer1 and xPer2) exhibit different temporal patterns of expression in the Xenopus retina, and that one (xPer2) is directly responsive to light and dopamine, it is not known whether this reflects the properties of period genes within photoreceptor oscillators or among distinct retinal cell populations. We addressed this by determining the cellular site of light and dopamine regulated xPer2 expression, and the diurnal expression of both xPer1 and xPer2 using in situ hybridization. Our data show that both xPer1 and xPer2 are expressed in most cell types in the retina, including inner nuclear neurons and ganglion cells. However, light and quinpirole, a dopamine agonist, increase xPer2 levels specifically in photoreceptors, and the effect of quinpirole, but not light, is blocked by pCPT-cAMP. Furthermore, antiphasic diurnal expression of xPer1 and xPer2 also occurs in photoreceptors. Our analysis does not provide insight into the near constitutive expression of period genes in the inner retina, but supports a model in which light- and dopamine regulated-xPer2 and rhythmic xPer1 play critical roles in entrainment and circadian oscillations within photoreceptors.