The recombinant production of therapeutic proteins for human diseases is currently the largest source of innovation in the pharmaceutical industry. The market growth has been the driving force on efforts for the development of new therapeutic proteins, in which transgenesis emerges as key component. The use of the transgenic animal platform offers attractive possibilities, residing on the low production costs allied to high productivity and quality of the recombinant proteins. Although many strategies have evolved over the past decades for the generation of transgenic founders, transgenesis in livestock animals generally faces some challenges, mainly due to random transgene integration and control over transgene copy number. But new developments in gene editing with CRISPR/Cas system promises to revolutionize the field for its simplicity and high efficiency. In addition, for the final approval of any given recombinant protein for animal or human use, the production and characterization of bioreactor founders and expression patterns and functionality of the proteins are technical part of the process, which also requires regulatory and administrative decisions, with a large emphasis on biosafety. The approval of two mammary gland-derived recombinant proteins for commercial and clinical use has boosted the interest for more efficient, safer and economic ways to generate transgenic founders to meet the increasing demand for biomedical proteins worldwide.
Apolipoprotein E (APOE=gene, apoE=protein) is a known factor regulating the
inflammatory response that may have regenerative effects during tissue recovery from
injury. We investigated whether apoE deficiency reduces the healing effect of
alanyl-glutamine (Ala-Gln) treatment, a recognized gut-trophic nutrient, during
tissue recovery after 5-FU-induced intestinal mucositis. APOE-knockout
(APOE-/-) and wild-type (APOE+/+) C57BL6J male and female
mice (N=86) were given either Ala-Gln (100 mM) or phosphate buffered saline (PBS) by
gavage 3 days before and 5 days after a 5-fluorouracil (5-FU) challenge (450 mg/kg,
via intraperitoneal injection). Mouse body weight was monitored daily. The 5-FU
cytotoxic effect was evaluated by leukometry. Intestinal villus height, villus/crypt
ratio, and villin expression were monitored to assess recovery of the intestinal
absorptive surface area. Crypt length, mitotic, apoptotic, and necrotic crypt
indexes, and quantitative real-time PCR for insulin-like growth factor-1 (IGF-1) and
B-cell lymphoma 2 (Bcl-2) intestinal mRNA transcripts were used to evaluate
intestinal epithelial cell turnover. 5-FU challenge caused significant weight loss
and leukopenia (P<0.001) in both mouse strains, which was not improved by Ala-Gln.
Villus blunting, crypt hyperplasia, and reduced villus/crypt ratio (P<0.05) were
found in all 5-FU-challenged mice but not in PBS controls. Ala-Gln improved
villus/crypt ratio, crypt length and mitotic index in all challenged mice, compared
with PBS controls. Ala-Gln improved villus height only in APOE-/- mice.
Crypt cell apoptosis and necrotic scores were increased in all mice challenged by
5-FU, compared with untreated controls. Those scores were significantly lower in
Ala-Gln-treated APOE+/+ mice than in controls. Bcl-2 and IGF-1 mRNA
transcripts were reduced only in the APOE-/--challenged mice. Altogether
our findings suggest APOE-independent Ala-Gln regenerative effects after 5-FU
challenge.
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