Study of the regulatory elements of the Ovalbumin gene promoter using CRISPR technology in chicken cells

Abstract Background Hormone-dependent promoters are very efficient in transgene expression. Plasmid-based reporter assays have identified regulatory sequences of the Ovalbumin promoter that are involved in response to estrogen and have shown that the deletion of the steroid-dependent regulatory element (SDRE) and negative regulatory element (NRE) leads to a steroid-independent expression of a reporter. However, the functional roles of these regulatory elements within the native genomic context of the Ovalbumin promoter have not been evaluated. Results In this study, we show that the negative effects of the NRE element on the Ovalbumin gene can be counteracted by CRISPR interference. We also show that the CRISPR-mediated deletion of SDRE and NRE promoter elements in a non-oviduct cell can lead to the significant expression of the Ovalbumin gene. In addition, the targeted knock-in of a transgene reporter in the Ovalbumin coding region and its expression confirms that the truncated promoter of the Ovalbumin gene can be efficiently used for an estrogen-independent expression of a foreign gene. Conclusions The methodology applied in this paper allowed the study of promoter regulatory sequences in their native nuclear organization

Integrating Omics and CRISPR Technology for Identification and Verification of Genomic Safe Harbor Loci in the Chicken Genome

Abstract 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

Enhanced cultivation of chicken primordial germ cells

Abstract The cultivation and expansion of chicken primordial germ cells (cPGCs) are 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 cPGCs. However, despite some success in the cultivation of cPGCs, 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. To this end, cPGCs were isolated from the embryonic blood of Hamburger–Hamilton (HH) stages 14–16 and cultured at various time points. The Growth properties and characteristics of these cells were evaluated in two different culture conditions (the defined or enriched medium) and their migratory properties were assessed after genetic engineering and injection into the vasculature of 2.5-day-old chicken embryos. The main finding of this study was that the use of an enriched medium (the defined medium with Knock-Out Serum Replacement; KOSR) resulted in improved growth properties of cPGCs originating from the Hubbard JA57 broiler compared to a defined medium. The ability to cultivate and expand cPGCs is crucial for the generation of both genetically engineered birds and breeds of interest from local or commercial origins. Therefore, these results highlight the importance of choosing an appropriate culture medium for cPGCs growth and expansion