Inserting random and site-specific changes into the genome of chickens.

Abstract

During the past decade, modifications to the chicken genome have evolved from random insertions of small transgenes using viral vectors to site-specific deletions using homologous recombination vectors and nontargeted insertions of large transgenes using phi-31 integrase. Primordial germ cells (PGC) and gonocytes are the germline-competent cell lines in which targeted modifications and large transgenes are inserted into the genome. After extended periods of in vitro culture, PGC retain their capacity to form functional gametes when reintroduced in vivo. Rates of stable germline modification vary from 1×10(-5) for nontargeted insertions to 1×10(-8) for targeted insertions. Following transfection, clonally derived cell lines are expanded, injected into Stage 13-15 Hamburger and Hamilton embryos, and putative chimeras are incubated to term in surrogate shells. Green fluorescent protein (GFP) is incorporated into transgenes to reveal the presence of genetically modified PGC in culture and the extent of colonization of the gonad during the first week posthatch. If the extent of colonization is adequate, cohorts of putative chimeras are reared to sexual maturity. Semen is collected and the contribution from donor PGC is estimated by evaluating GFP expression using flow cytometry and PCR. The most promising candidates are selected for breeding to obtain G1 heterozygote offspring. To date, this protocol has been used to (1) knockout the immunoglobulin heavy and light chain genes and produce chickens lacking humoral immunity, (2) insert human V genes and arrays of pseudo V genes into the heavy and light immunoglobulin loci to produce chickens making antibodies with human V regions, (3) insert GFP into nontargeted locations within the genome to produce chickens expressing GFP, and (4) insert Cre recombinase into the genome to produce chickens that excise sequences of DNA flanked by loxP sites.