Efficient production of recombinant human adiponectin in egg white using genome edited chickens

Abstract

The prevalence of obesity-related metabolic diseases caused by insulin resistance is rapidly increasing worldwide. Adiponectin (ADPN), a hormone derived from adipose tissue, is a potential therapeutic agent for insulin resistance. Chickens are considered efficient bioreactors for recombinant protein production because they secrete large amounts of high-concentration proteins from the oviduct. Additionally, chickens express high levels of high-molecular-weight (HMW) ADPN, which is considered the active form in the body. Therefore, in this study, a gene-targeted chicken model was produced in which the gene encoding human ADPN was inserted into Ovalbumin (OVA) using the CRISPR/Cas9 system, and the characteristics of the resulting recombinant ADPN protein were evaluated. As a result, human ADPN was expressed in G1 hen oviducts and egg whites of OVA ADPN knock-in (KI) chickens. The concentration of ADPN in egg white ranged from 1.47 to 4.59 mg/mL, of which HMW ADPN accounted for ∼29% (0.24–1.49 mg/mL). Importantly, egg white-derived ADPN promoted expression of genes related to fatty acid oxidation and activated the 5′-AMP-activated protein kinase (AMPK) signaling pathway in muscle cells. In summary, the OVA gene-targeted chicken bioreactor proved to be an advantageous model for production of human ADPN, and the resulting protein was of sufficient quantity and efficacy for industrial use.

Lipofection with Lipofectamine™ 2000 in a heparin‐free growth medium results in high transfection efficiency in chicken primordial germ cells

“…Although we have knocked‐in an EGFP construct (about 1 kb), the increased lipofection efficiency will facilitate the knock‐in of large gene fragments of several kb, which has been previously achieved. [ 2–5,15 ] However, in this study, knocked‐in PGCs only accounted for approximately 4% of all cells, even with the HMEJ strategy. Future studies must consider enriching only vector‐transfected cells, or combining optimized lipofection with other knock‐in strategies.…”

“…Furthermore, chickens have been proven valuable as bioreactors via genome editing. [2][3][4][5] However, applying genome editing in birds, including chickens, has not advanced compared to other animals.…”

Sustainable production of multimeric and functional recombinant human adiponectin using genome-edited chickens

“…To analyze hADPN present in egg white (EW) from OVA ADPN knock-in (KI) chickens, western blotting procedures were performed as previously described [ 29 ]. Briefly, for non-reducing conditions, samples were mixed with an equal volume of native sample buffer (BioRad, Hercules, CA, USA).…”

“…Furthermore, hyperglycemia counteracts the suppressive effect of hyperinsulinemia on plasma ADPN levels in humans, providing chickens with a unique advantage in ADPN levels due to their naturally occurring hyperglycemic and insulin-resistant state [ 26 – 28 ]. Recently, functional hADPN, predominantly in multimeric forms, was successfully produced using chickens through ovalbumin ( OVA )-targeted clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing [ 29 ].…”

Strategies for the Generation of Gene Modified Avian Models: Advancement in Avian Germline Transmission, Genome Editing, and Applications

“…In addition, gene encoding adiponectin (ADPN), a hormone derived from adipose tissue that can be used to treat insulin resistance, was precisely integrated into the Ovalbumin ( OVA ) by CRISPR/Cas9 system. The gene-edited chicken expressed high amount of high-molecular-weight (HMW) ADPN, considered to be a more active form [ 184 ]. Recently, the GFP gene was inserted into chicken ovalbumin ( OVA ) gene and system for evaluating protein production in a chicken bioreactor using young chicks was established.…”