Obtaining chicken primordial germ cells used for gene transfer: in vitro and in vivo results

Abstract

Recently, several attempts have been made to create a generation of transgenic chickens via chimeric intermediates produced by primordial germ cells (PGCs) transfer. This study aimed to compare the influences of different chicken PGCs isolated from circulating blood (bPGCs) or gonads (gPGCs), purification (ACK, Percoll or trypsin) and transfection methods (electroporation or lipofection) on the expression of transgenes in vitro and the migration of modified donor cells to the recipient gonads. The highest average frequency of pEGFP-N1 plasmid-transfected bPGCs (75.8 %) was achieved with Percoll density gradient centrifugation and electroporation. After ammonium chloride-potassium (ACK) treatment and lipofection, in vitro transgene expression was only detected in 35.2 % of bPGCs. Chimeric chickens were produced from these purified, transfected and cultured cells, and the transgene was detected in the gonads of 44 and 42 % of the recipient embryos that had been injected with bPGCs and gPGCs, respectively. These data confirmed that the combination of PGC purification via Percoll centrifugation and electroporation was an effective method for producing transgenic chickens. Subsequently, we used this method with expression vectors for gene hIFNα 2a/hepatitis B virus surface antigen (HBsAg) under the control of the ovalbumin promoter to generate G0 transgenic chickens. Consequently, we observed that 4.9 % of the hens and 3.5 % of the roosters carried the hIFNα 2a gene, whereas 16.7 % of the hens and 2.4 % of the roosters carried the HBsAg gene, thus undisputedly confirming the exceptional effectiveness of the applied methods.

In ovodelivery of various biological supplements, vaccines and drugs in poultry: current knowledge

“…It was initially incepted in the 1990s in the USA to automatically vaccinate chicks against Marek's disease and Gumboro disease in poultry . Since then, it has been used for a much wider application in order to stimulate embryonic development, immunological response, and beneficial probiotic bacteria . Casas‐Perez and Edens found that the administration of probiotics (lactic acid bacteria) via the in ovo technique, should be done on the 18th day of incubation.…”

CRISPR/Cas9 gene editing in a chicken model: current approaches and applications

“…In this stage, PGCs can be commonly acquired through gonad isolation and trypsinization. PGCs can transmit genetic information to next generations and are thus ideal for creating transgenic or gene-edited chickens (Bednarczyk et al 2002;Bednarczyk et al 2003; Van de Lavoir et al 2006;Naito 2015;Chojnacka-Puchta et al 2015;Sawicka et al 2015;Oishi et al 2016;Dimitrov et al 2016). However, PGCs are characterized by their low ability to induce effective and persistent transfection.…”

PAZ Domain is Critical for PIWIL1 Function During Spermatogenesis in Chicken

“…Germline cells identify, cell culture, and induction with retinoic acid (RA) PGC colonies were identi ed using a mouse anti-chicken c-KIT antibody (1:50) as a primary antibody and goat-anti-mouse FITC-conjugated IgM (1:50) as a secondary antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA). The identi ed PGCs were subjected to periodic acid Schiff (PAS) staining as previously described [12]. Brie y, PGCs were xed in 4% paraformaldehyde for 5 min, rinsed twice with 1 × PBS (Hyclone, South Logan, UT, USA), immersed in periodic acid solution for 5 min, washed with 1 × PBS, immersed in Schiff's solution for 15 min, washed twice in 1 × PBS, and observed under an inverted microscope.…”