Interspecific Germline Transmission of Cultured Primordial Germ Cells

In birds, the primordial germ cell (PGC) lineage separates from the soma within 24 h following fertilization. Here we show that the endogenous population of about 200 PGCs from a single chicken embryo can be expanded one million fold in culture. When cultured PGCs are injected into a xenogeneic embryo at an equivalent stage of development, they colonize the testis. At sexual maturity, these donor PGCs undergo spermatogenesis in the xenogeneic host and become functional sperm. Insemination of semen from the xenogeneic host into females from the donor species produces normal offspring from the donor species. In our model system, the donor species is chicken (Gallus domesticus) and the recipient species is guinea fowl (Numida meleagris), a member of a different avian family, suggesting that the mechanisms controlling proliferation of the germline are highly conserved within birds. From a pragmatic perspective, these data are the basis of a novel strategy to produce endangered species of birds using domesticated hosts that are both tractable and fecund

Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds

Growth curves of CRISPR progeny.

<p>Eight heterozygous IgH KO6B females and eight control females were weighed weekly for 8 weeks.</p

CRISPR-mediated targeting of IgH KO6B in PGCs.

<p>A. Detailed diagram of the IgH locus. The 122 bp sequence between the 5’ and 3’ homology regions in IgH KO6B, used to design the gRNAs, is shown at top. The locations of gRNAs 1–4 are indicated with blue lines above the sequence and the protospacer adjacent motifs (PAM) indicated with red lines. The repair vector IgH KO6B (below) contains 5’ and 3’ homology regions (HR) in yellow, a single loxP site (blue arrowhead), and a hygromycin selection cassette (orange). The locations of the primer binding sites for the 5’ and 3’ targeting assays are shown as black arrows. The downstream selectable markers in the JH-KO consist of floxed EGFP (green box) and puro gene (blue box), and a promoterless neo gene in opposite orientation (pink box). The loxP sites are blue arrowheads. B. The 5’ targeting assay performed on independent, non-clonal cell populations obtained from co-transfection of the four different gRNAs into 472–138 cells along with Cas9 and IgH KO6B. For each gRNA transfection, 3 hygromycin-resistant populations were analyzed. The positive control (+) was a DT40 cell line that contained a knockout of the functional V region [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154303#pone.0154303.ref022" target="_blank">22</a>] and the negative control (-) was the parental IgH KO6B plasmid. C. The 5’ and 3’ targeting assays performed on 9 independent clones obtained with gRNA2 (there were 12 clones, but clones 4, 7 and 12 grew more slowly and were not tested at this time). Variation in band intensity is likely to be from variation in the template gDNA amount, since the number of cells harvested was not normalized. The negative control (-) was genomic DNA from a JH-KO transgenic bird, and the positive control (+) was a pool of cells (G2) from the gRNA2 experiment in B. NT, no template control. D. The same 5’ and 3’ targeting assays performed on EGFP+ birds obtained from breeding cell line 1783–10 chimeras to wild type.</p

Germline transmission rates of chimeras made with CRISPR-modified PGCs.

<p>Germline transmission rates of chimeras made with CRISPR-modified PGCs.</p

Strategy for CRISPR targeting.

<p>A. Diagram of the chicken IgH locus present in PGC line 472–138 used for CRISPR targeting. The IgH locus contained a previously obtained knockout of the JH gene segment (JH-KO), between the D cluster and the constant regions (only Cμ is shown), which was replaced with a selectable marker cassette. gRNAs 1 through 4 were designed to target a region upstream of the single functional VH region (indicated with an arrow), and gRNA5 was designed to target the EGFP gene. B. PGCs of line 472–138 were transiently transfected with a construct containing Cas9 or Cas9/gRNA5, specific for EGFP. After 9 days in culture, the cells were analyzed by flow cytometry for loss of green fluorescence.</p

Cre recombination of CRISPR-targeted loxP site.

<p>A. Diagram of the targeted IgH locus before and after Cre recombination. A forward primer upstream of the CRISPR-targeted loxP site was used with two different reverse primers downstream of the loxP site in the JH-KO cassette. In the non-recombined allele, the forward and reverse primers are separated by about 28kb on the chromosome. After Cre recombination, a single loxP site and the promoterless neo gene remain, and the primers are either 1.6 or 2kb apart, which amplifies readily. B. PCR of recombined cells. Cre +: gDNA template from 1783–9 cells transfected with Cre; Cre -, parental 1783–9 cells; JH-KO, gDNA from a heterozygous JH-KO bird; NTC, no template control.</p

Colonization of xenogeneic testis (Left panel) GFP positive chicken primordial germ cells in culture on irradiated BRL feeder cells.

<p>(Right panel) To evaluate the incorporation of chicken GFP positive cells into the gonads of guinea fowl, testes were retrieved, fixed in 4% paraformaldehyde and processed for cryosectioning. GFP expressing cells are present only in the seminiferous tubules suggesting that they have differentiated into spermatogonia. The absence of GFP-expressing cells in the interstitial tissue is consistent with the exclusive commitment of PGCs to the germline. Sizebar denotes 100 µm</p

Pedigree analysis of offspring.

<p>PCR amplification of genomic DNA from combs of chicks, blood from their wild-type mothers, semen from their interspecific chimeric fathers and the 169.4 PGC and 167.2 cell lines that were incorporated into the testis of the interspecific chimeric males. The GFP locus in the 169.4 and 167.2 is heterozygous and therefore, approximately one half of the offspring from the injected PGCs inherit the locus and express GFP. Top panel, offspring from the 169.4 cell line. Bottom panel; offspring from the 167.2 cell line.</p

Evaluation of sperm by flow cytometry and production of GFP-expressing and naked neck offspring from interspecific male guinea fowl chimeras.

<p>The GFP and naked neck loci are heterozygous and therefore, one half of the offspring from the injected PGCs inherit the expressed alleles at these loci. The high rates of fertility indicate that the testis is highly colonized by chicken PGCs in some of the interspecific male guinea fowls. Hybrids appear in the offspring of roosters with lower rates of germline transmission. GFP transmission is calculated from all embryos evaluated including embryos at D7 and chicks at hatch. Naked neck transmission was evaluated in hatched chicks and the hybrid percentage was calculated as a percentage of all offspring including both chicks and hybrids.</p