Generating biopharmaceuticals in genetically engineered bioreactors continues to reign supreme. Hence, genetically engineered birds have attracted considerable attention from the biopharmaceutical industry. Fairly recent genome engineering methods have made genome manipulation an easy and affordable task. In this review, we first provide a broad overview of the approaches and main impediments ahead of generating efficient and reliable genetically engineered birds, and various factors that affect the fate of a transgene. This section provides an essential background for the rest of the review, in which we discuss and compare different genome manipulation methods in the pre-CRISPR and CRISPR era in the field of avian genome engineering.

The ability and efficiency of targeted nucleases to perform sequence replacements or insertions into the genome are limited. This limited efficiency for sequence replacements or insertions can be explained by the dependency on DNA repair pathways, the possibility of cellular toxicity, and unwanted activation of proto-oncogenes. The piggyBac (PB) transposase uses a very efficient enzymatic mechanism to integrate DNA fragments into the genome in a random manner. In this study, we fused an RNA-guided catalytically inactive Cas9 (dCas9) to the PB transposase and used dual sgRNAs to localize this molecule to specific genomic targets. We designed and used a promoter/reporter complementation assay to register and recover cells harboringspecific integrations, where only by complementation upon correct genomic integration, the reporter can be activated. Using an RNA-guided piggyBac transposase and dual sgRNAs, we were able to achieve site-directed integrations in the human ROSA26 safe harbor region in 0.32% of cells. These findings show that the methodology used in this study can be used for targeting genomic regions. An application for this finding could be in cancer cells to insert sequences into specific target regions that are intended to be destroyed, or to place promoter cargos behind the tumor suppressor genes to activate them. K E Y W O R D SCRISPR-associated protein 9, gene insertion, Insertional mutagenesis, piggyBac transposase, transposases 2 of | GOSHAYESHI Et Al.

Background One of the most prominent questions in the field of transgenesis is ‘Where in the genome to integrate a transgene?’. Escape from epigenetic silencing and promoter shutdown of the transgene needs reliable genomic safe harbor (GSH) loci. Advances in genome engineering technologies combined with multi-omics bioinformatics data have enabled rational evaluation of GSH loci in the host genome. Currently, no validated GSH loci have been evaluated in the chicken genome. Results Here, we analyzed and experimentally examined two GSH loci in the genome of chicken cells. To this end, putative GSH loci including chicken HIPP-like (cHIPP; between DRG1 and EIF4ENIF1 genes) and chicken ROSA-like (cROSA; upstream of the THUMPD3 gene) were predicted using multi-omics bioinformatics data. Then, the durable expression of the transgene was validated by experimental characterization of continuously-cultured isogenous cell clones harboring DsRed2-ΔCMV-EGFP cassette in the predicted loci. The weakened form of the CMV promoter (ΔCMV) allowed the precise evaluation of GSH loci in a locus-dependent manner compared to the full-length CMV promoter. Conclusions cHIPP and cROSA loci introduced in this study can be reliably exploited for consistent bio-manufacturing of recombinant proteins in the genetically-engineered chickens. Also, results showed that the genomic context dictates the expression of transgene controlled by ΔCMV in GSH loci.

Our knowledge of mechanisms involved in the meiosis of chicken germ cells is very limited. In mammalian fetal ovaries, the onset of meiosis is dependent on retinoic acid and subsequent upregulation of the Stra8 gene. To clarify the mechanism of meiotic initiation in chicken germ cells, we investigated the role of Cyp26b1, a retinoic acid‐degrading enzyme. The Cyp26b1‐inhibitor, ketoconazole was used to treat the ex vivo‐cultured stage 36 gonads/mesonephroi. Then, the progression of meiosis was studied by histological and immunohistochemical analysis and the level of the transcript for Stra8 was evaluated by a quantitative reverse transcription‐polymerase chain reaction in individual ketoconazole‐treated gonads after 6 days in culture. The results revealed that meiosis was induced in both testes and right ovary upon inhibition of Cyp26b1 in the ex vivo‐cultured gonads, despite downregulation of Stra8 messenger RNA in the treated gonads. Also, meiosis was observed only when mesonephros was cultured alongside the left ovary. These findings demonstrate that in chicken, Stra8 is not the only factor for the entrance into meiosis, and Cyp26b1 and mesonephros play critical regulatory roles for the sex‐specific timing of meiotic initiation in birds.

Background The cultivation and expansion of chicken primordial germ cells (cPGCs) is of critical importance for both biotechnological applications and the management of poultry genetic biodiversity. The feeder-free culture system has become the most popular approach for the cultivation and expansion of chicken primordial germ cells. However, despite some success in the cultivation of PGCs, the reproducibility of culture conditions across different laboratories remains a challenge. This study aimed to compare two defined and enriched media for the growth of cPGCs originating from the Hubbard JA57 broiler.Methods To this end, cPGCs were isolated from the embryonic blood of 14–16 Hamburger–Hamilton (HH) stages and cultured at various time points. The Growth properties and characteristics of these cells were evaluated in two different culture conditions and their migratory properties were assessed after genetic engineering and injection into the vasculature of 2.5-day-old chicken embryos.Results The main finding of this study was that the use of an enriched medium resulted in improved growth properties of PGCs compared to a defined medium.Conclusions The ability to cultivate and expand cPGCs is crucial for various key approaches and a robust supportive medium can help to accelerate the generation of both genetically engineered birds in avain transgenesis approach and breeds of interest from local or commercial origins. Therefore, these results highlight the importance of choosing an appropriate culture medium for cPGC growth and expansion.