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

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

Expression and signaling in DT40 cells with immunoglobulin knockouts and chimeric immunoglobulin insertions.

<p>Wild-type cells (DT40 wt), V_L knockout (<i>Igl^KO</i>), V_H knockout (<i>Igh^KO</i>) and V_L-V_H double knockout cells (<i>Igl^KO, Igh^KO</i>) as well as huV_K insertion (<i>Igl^huVK</i>), huV_H insertion (<i>Igh^huVH</i>) and huV_K-huV_H (<i>Igl^huVK, Igh^huVH</i>) double insertion cell lines were analyzed for expression and signaling of immunoglobulin receptors. a) 1×10⁶ cells were lysed and immunoglobulin heavy chain expression determined by Western blotting using goat-anti-chicken-IgM-AP and immunoglobulin light chain expression by rabbit-anti-chicken-IgY-AP. Mouse-anti-β-actin followed by goat-anti-mouse-AP was used to detect β-actin. b) The cell lines from above and <i>Igl^huVK</i> cells with a stop codon (<i>Igl^huVK-Stop</i>) in CDR1 cultured for four weeks were stained with mouse-anti-chicken-IgM followed by goat-anti-mouse-Ig-Cy5. All cell lines except wild type express eGFP from the selectable marker cassette used in the knockouts. Fluorescence signal was visualized using a Beckman Coulter FC-500. c) 1×10⁶ wild type DT40 cells, non-green <i>Igl^KO, Igh^KO</i> cells and non-green <i>Igl^huVK, Igh^huVH</i> DT40 cells were labeled with FLUO-4-AM and incubated with 10 µg/ml goat-anti-chicken-IgM starting from the time point indicated by arrows. The change in fluorescence intensity was measured for a total of 300 sec using a Beckton Dickinson LSRII Fortessa. One of three representative experiments is shown. d) The same cell lines (DT40 wt grey, <i>Igl^KO, Igh^KO</i> red, <i>Igl^huVK, Igh^huVH</i> blue) were stained with goat-anti-human-kappa-RPE. Fluorescence was measured using a Beckman Coulter FC-500.</p

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

<p>The human SynVH-B pseudogenes SynVH21 to SynVH37 were aligned with functional huV_H using Lasergene (DNAStar Inc.,Madison, USA). CDRs are marked by upright dashed lines. The arrow indicates position of the HpaI site and stop codon inserted in a) CDR1 or b) CDR3.</p

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

Integrase-mediated insertion of human V_L and V_H to replace the corresponding chicken genes.

<p>The <i>Igl^KO</i> and <i>Igh^KO</i> cell lines, as well as the <i>Igl^KO, Igh^KO</i> line, were used to insert human V_K or V_H into the chicken loci. Co-transfection of ΦC31 integrase and the shown a) huV_K or b) huV_H constructs resulted in recombination of the attP and attB sites leading to an insertion of the human V constructs and creating attL and attR sites. A β-actin promoter was integrated in front of the neomycin (neo) or blasticidin (Bsr) gene, and cells were selected with the indicated drug for stable integration of the huV_K or huV_H. In the case of huV_K insertion into the chicken <i>Igl^KO</i> single knockout, the selectable marker cassette was the same as for the heavy chain locus shown in b). c) To test for proper integration of the human genes, genomic DNA was isolated and a construct-specific PCR was performed with the indicated primers (primers are indicated by black arrowheads with primer orientation: VK+VH 5′, primers 12 and 9; VK 5′ Bsr, primers 8 and 9; VK 3′, primers 10 and 11; VH 3′, primers 13 and 14). Primers are placed on the 5′ and 3′ side of the integration showing the correct integration versus the knock out or wild type DT40 cell lines. β-actin was used as a quality control for the genomic DNA. Scale bar equals 1 kb.</p

Alignment of the SynVK-12 pseudogene array with functional huV_K.

<p>The human SynVK-12 pseudogenes SynVK1 to SynVK12 were aligned with functional huV_K using Lasergene (DNAStar Inc.,Madison, USA). Complementarity determining regions (CDR) 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

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