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

Regulation and Genetics of Amino Acid Transport

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75377/1/j.1749-6632.1985.tb14892.x.pd

Harnessing gene conversion in chicken B cells to create a human antibody sequence repertoire.

Transgenic chickens expressing human sequence antibodies would be a powerful tool to access human targets and epitopes that have been intractable in mammalian hosts because of tolerance to conserved proteins. To foster the development of the chicken platform, it is beneficial to validate transgene constructs using a rapid, cell culture-based method prior to generating fully transgenic birds. We describe a method for the expression of human immunoglobulin variable regions in the chicken DT40 B cell line and the further diversification of these genes by gene conversion. Chicken VL and VH loci were knocked out in DT40 cells and replaced with human VK and VH genes. To achieve gene conversion of human genes in chicken B cells, synthetic human pseudogene arrays were inserted upstream of the functional human VK and VH regions. Proper expression of chimeric IgM comprised of human variable regions and chicken constant regions is shown. Most importantly, sequencing of DT40 genetic variants confirmed that the human pseudogene arrays contributed to the generation of diversity through gene conversion at both the Igl and Igh loci. These data show that engineered pseudogene arrays produce a diverse pool of human antibody sequences in chicken B cells, and suggest that these constructs will express a functional repertoire of chimeric antibodies in transgenic chickens

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

Creating huV_H diversity by gene conversion using a human heavy chain pseudogene array with fully synthetic CDRs.

<p>The chicken V_H was replaced by a huV_H including 7 human pseudogenes with fully synthetic CDR sequences (the SynVH-A7 array). The inserted huV_H had a stop codon and a HpaI restriction enzyme site in CDR3 so that there was no IgM expression unless the stop codon was repaired due to gene conversion. DT40 cells were cultured for four weeks after insertion and afterwards analyzed by sequencing for possible gene conversion events, deletions and point mutations. Gene conversion events for the stop in CDR3 are shown. The length of the line showing the gene conversion events corresponds with the actual length of the gene conversion event observed. Frequency of every event is shown in bold. Every change in the parental sequence was counted as individual event.</p

Creating huV_H diversity by gene conversion using a human heavy chain pseudogene array with single amino acid changes in the CDRs.

<p>The chicken V_H was replaced by a huV_H including 17 human synthetic pseudogenes with single amino acid changes in the CDR sequences (the SynVH-B array). The inserted huV_H had a stop codon and a HpaI restriction enzyme site in CDR1 or CDR3 so that there was no IgM expression unless the stop codon was repaired due to gene conversion. DT40 cells were cultured for four weeks after insertion and afterwards analyzed by sequencing for possible gene conversion events, deletions and point mutations. Gene conversion events for the stop in CDR1 are shown in a) and b). Gene conversion events for the stop in CDR3 are shown in c). No pseudogene name is indicated over the line in CDR3 in c) because the sequences could have been derived from every pseudogene except 22 and 30. The length of the line showing the gene conversion events corresponds with the actual length of the gene conversion event observed. Frequency of every event is shown in bold. Every change in the parental sequence was counted as individual event.</p

Creating huV_K diversity by gene conversion using a human light chain pseudogene array with naturally occurring CDRs, AID optimization and framework changes.

<p>The chicken V_L was replaced by a huV_K including 16 human synthetic pseudogenes containing naturally occurring CDR sequences from an EST database (NCBI), the SynVK-C array. In addition the pseudogenes and the functional V_L were AID optimized. Some of the pseudogenes also differ in their framework regions compared to the functional huV_K. The inserted huV_K had a stop codon and a HpaI restriction enzyme site in CDR1 or CDR3 so that there was no IgM expression unless the stop codon was repaired due to gene conversion. DT40 cells were cultured for four weeks after insertion and afterwards analyzed by sequencing for possible gene conversion events, deletions and point mutations. Gene conversion events for the stop in CDR1 are shown in a) and b). Gene conversion events for the stop in CDR3 are shown in c). The length of the line showing the gene conversion events corresponds with the actual length of the gene conversion event observed. Frequency of every event is shown in bold at left. The arrows under the pseudogenes and the functional V in b) show the orientation of the genes. Every change in the parental sequence was counted as individual event.</p

Alignment of the SynVK-C pseudogene array with functional huV_K.

<p>The human SynVK-C pseudogenes SynVK20 to SynVK35 were aligned with functional huV_K using Lasergene (DNAStar Inc.,Madison, USA). CDRs are marked by upright dashed lines. The arrow indicates the position of the HpaI site and stop codon inserted in a) CDR1 or b) CDR3.</p

Alignment of the SynVH-A7 pseudogene array with functional huV_H.

<p>The human SynVH-A7 pseudogenes SynVH14 to SynVH20 were aligned with functional huV_H using Lasergene (DNAStar Inc.,Madison, USA). CDRs are marked by upright dashed lines. The arrow indicates the position of the HpaI site and stop codon inserted in CDR3.</p