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

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

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

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

<p>The chicken V_L was replaced by a huV_K including 12 human synthetic pseudogenes with single amino acid changes in the CDR sequences (the SynVK-12 array). 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) and d). In diagrams a) and c), the length of the line showing the gene conversion events corresponds with the actual length of the gene conversion event observed, and the number above the line indicates the pseudogene name. Frequency of every event is shown in bold at left. The arrows under the pseudogenes and the functional V in c–d) show the orientation of the genes. Every change in the parental sequence was counted as an individual event.</p

Knockout of the chicken V_L and V_H genes.

<p>Chicken V_L and V_H loci are shown and the homology regions of the knockout constructs flanking the deleted region are marked by dotted lines. Targeting constructs for the V_L and V_H in chicken DT40 cells are shown. a) For the V_L single knockout, the knockout construct consists of a β-actin-eGFP (eGFP), a CAG-puromycin (puro) selectable marker cassette and the 5′ and 3′ homology regions. To create the double knockout of V_L and V_H, an alternative targeting construct for V_L has a β-actin-hygromycin (hygro) selectable marker cassette. The homology regions are the same for both V_L knockout constructs. b) For the V_H knockout, the targeting construct consists of a 5′ and 3′ homology region, a β-actin-eGFP and a CAG-puromycin selectable marker cassette. In all constructs, the selectable markers are flanked by the HS4 insulator from chicken beta-globin (H), and loxP sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080108#pone.0080108-RecillasTarga1" target="_blank">[39]</a>. Downstream of the 3′ loxP site a promoterless neomycin (neo) or blasticidin (Bsr) gene in opposite orientation together with an attP site for targeted insertion is included. c) Knockout of the V_L and V_H was detected with gene specific primers on the 5′ side as well as with primers in the deleted region (black arrow heads: VL 5′, primers 1 and 2; VL deletion region, primers 3 and 4; VH 5′, primers 5 and 2; VH deleted region, primers 6 and 7). β-actin served 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