Silencing Acetyl-CoA carboxylase A (ACACA) and Sterol Regulatory Element-binding Protein 1 (SREBP1) Genes Through RNAi Reduce Cholesterol Content in Serum and Eggs of Transgenic Chicken

Cholesterol is synthesized in chicken through de novo lipid biosynthetic pathway where two most important genes viz. SREBP1 and ACACA play immense role. To minimize cholesterol synthesis, RNAi approach was adopted and accordingly, we developed transgenic chicken possessing ACACA and SREBP1 shRNA constructs, which showed lower level of ACACA and SREBP1 in serum. The serum total cholesterol and LDL cholesterol was significantly (P<0.05) lower by 26.8 and 31.3%, and 56.3 and 26.4%, respectively in ACACA and SREBP1 transgenic birds compared to the control. The egg total cholesterol and LDL cholesterol content was significantly (P<0.05) lower in both ACACA and SREBP1 transgenic birds by 14.3 and 13.2%, and 10.3 and 13.6%, respectively compared to the control. It is concluded that the protocol was perfected to develop transgenic chicken through RNAi for knocking down the expression of ACACA and SREBP1 proteins, which minimized the cholesterol and triglycerides contents in serum and eggs.

Production of Transgenic Chimeric Chicken from Cryopreserved Primordial Germ Cells and its Validation by Developing shRNA Transgenic Chicken Chimera

Primordial germ cells (PGCs) are precursors of gametes in birds. For ex-situ conservation and production of transgenic birds, there is a limitation for preservation of oocytes in birds as compared to other mammalian species. To overcome those limitations, PGCs have been used as candidate cells, which have been cryopreserved and manipulated and used as to produce transgenic birds. In this study, cryo-preserved PGCs were used to produce transgenic birds. The protocol for production of transgenic birds with cryo-preserved PGCs was developed and the success rate for production of transgenics 16.7% in the protocols established in the study. The same gene transfer protocol through PGCs was validated by transferring shRNA molecule of SREBP-1 gene to the host genome to produce transgenic chimeric birds and the success rate for production of transgenic chimeric chicken was 40%. Finally, it is concluded that a standard protocol for ex-situ conservation of birds through PGCs and production of transgenic birds from cryo-preserved PGCs and knock down birds from PGCs were developed. It may be suggested that these protocols for resurrecting live birds from cryo-preserved PGCs may be applied as model for protecting the endangered birds from their extinction.

In Vivo Inhibition of Marek’s Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4

Marek’s disease (MD), caused by MD herpesvirus (MDV), is an economically important disease in chickens. The efficacy of the existing vaccines against evolving virulent stains may become limited and necessitates the development of novel antiviral strategies to protect poultry from MDV strains with increased virulence. The CRISPR/Cas9 system has emerged as a powerful genome editing tool providing an opportunity to develop antiviral strategies for the control of MDV infection. Here, we characterized Tol2 transposon constructs encoding Cas9 and guide RNAs (gRNAs) specific to the immediate early infected-cell polypeptide-4 (ICP4) of MDV. We generated transgenic chickens that constitutively express Cas9 and ICP4-gRNAs (gICP4) and challenged them via intraabdominal injection of MDV-1 Woodlands strain passage-19 (p19). Transgenic chickens expressing both gRNA/Cas9 had a significantly reduced replication of MDV in comparison to either transgenic Cas9-only or the wild-type (WT) chickens. We further confirmed that the designed gRNAs exhibited sequence-specific virus interference in transgenic chicken embryo fibroblast (CEF) expressing Cas9/gICP4 when infected with MDV but not with herpesvirus of turkeys (HVT). These results suggest that CRISPR/Cas9 can be used as an antiviral approach to control MDV infection in chickens, allowing HVT to be used as a vector for recombinant vaccines.

Production of Recombinant Monoclonal Antibodies in the Egg White of Gene-Targeted Transgenic Chickens

Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4–1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs.

HMEJ-mediated efficient site-specific gene integration in chicken cells

Background The production of transgenic chicken cells holds great promise for several diverse areas, including developmental biology and biomedical research. To this end, site-specific gene integration has been an attractive strategy for generating transgenic chicken cell lines and has been successfully adopted for inserting desired genes and regulating specific gene expression patterns. However, optimization of this method is essential for improving the efficiency of genome modification in this species. Results Here we compare gene knock-in methods based on homology-independent targeted integration (HITI), homology-directed repair (HDR) and homology mediated end joining (HMEJ) coupled with a clustered regularly interspaced short palindromic repeat associated protein 9 (CRISPR/Cas9) gene editing system in chicken DF-1 cells and primordial germ cells (PGCs). HMEJ was found to be a robust and efficient method for gene knock-in in chicken PGCs. Using this method, we successfully labeled the germ cell specific gene DAZL and the pluripotency-related gene Pou5f3 in chicken PGCs through the insertion of a fluorescent protein in the frame at the 3′ end of the gene, allowing us to track cell migration in the embryonic gonad. HMEJ strategy was also successfully used in Ovalbumin, which accounts for more than 60% of proteins in chicken eggs, suggested its good promise for the mass production of protein with pharmaceutical importance using the chicken oviduct system. Conclusions Taken together, these results demonstrate that HMEJ efficiently mediates site-specific gene integration in chicken PGCs, which holds great potential for the biopharmaceutical engineering of chicken cells.