Location: Food and Feed Safety Research
Project Number: 3091-32000-037-011-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 3, 2025
End Date: Jul 2, 2027
Objective:
The objective of this research is to generate chicken primordial germ cells resistant to Salmonella colonization. These will be used to generate chicken lines with inherent Salmonella resistance.
Approach:
Guide Sequences and CRISPR-Cas9: Guide sequences are designed with 20 base-pairs complementarity to the target sequence, with appropriate proximity to a PAM site (required for cleavage to occur), and a suitable hairpin sequence for interaction with Cas9. For each candidate gene, we will design three unique pairs of guide RNAs bracketing the target genes. The primers are cloned into a plasmid carrying the Crispr/Cas9 gene and then transfected into PGCs. Typically, at least one of the three successfully knocks-out the target.
PGC Preparation: PGC are prepared by isolation of blood from Stage 15 or 16 (Hamilton and Hamburger stages) embryos and grown in culture media supplemented with Fibroblast Growth Factor 2 and Activin A. Cells are grown until a concentration of 4x105 cells/ml and then frozen in liquid nitrogen for long-term storage.
PGC Clone Selection: Plasmid encoding the guide sequences will be mixed with the Cas9 encoding plasmid and transfected into PGC cells by electroporation. The Cas9 plasmid also encodes a puromycin resistance gene which allows for selection of cells that have taken up the plasmid. Subsequently individual clones are picked, expanded and then tested by PCR to identify clones with the correct deletion.