Alkaline Phosphatase

EXAMPLE 23

One llama (No. 149) was immunized with 6 boosts (100 or 50 μg/dose at weekly intervals) of R&D Systems (Minneapolis, MN, US) Cat #1224-PL, which is the ectodomain of human PD-L2 (rhPDL2-Fc), formulated in Titermax Gold (Titermax USA, Norcross, GA, US), according to standard protocols. At week 4, sera were collected to define antibody titers against PD-L2 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were coated with rhPDL2-Fc. After blocking and adding diluted sera samples, the presence of anti-PD-L2 Nanobodies was demonstrated by using rabbit anti-llama immunoglobulin antiserum and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The titer exceeded 16000.

Example 3

We generated and analyzed a collection of 14 early-passage (passage ≤9) human pES cell lines for the persistence of haploid cells. All cell lines originated from activated oocytes displaying second polar body extrusion and a single pronucleus. We initially utilized chromosome counting by metaphase spreading and G-banding as a method for unambiguous and quantitative discovery of rare haploid nuclei. Among ten individual pES cell lines, a low proportion of haploid metaphases was found exclusively in a single cell line, pES10 (1.3%, Table 1B). We also used viable FACS with Hoechst 33342 staining, aiming to isolate cells with a DNA content corresponding to less than two chromosomal copies (2c) from four additional lines, leading to the successful enrichment of haploid cells from a second cell line, pES12 (Table 2).

Two individual haploid-enriched ES cell lines were established from both pES10 and pES12 (hereafter referred to as h-pES10 and h-pES12) within five to six rounds of 1c-cell FACS enrichment and expansion (FIG. 1C (pES10), FIG. 5A (pES12)). These cell lines were grown in standard culture conditions for over 30 passages while including cells with a normal haploid karyotype (FIG. 1D, FIG. 5B). However, since diploidization occurred at a rate of 3-9% of the cells per day (FIG. 1E), cell sorting at every three to four passages was required for maintenance and analysis of haploid cells. Further, visualization of ploidy in adherent conditions was enabled by DNA fluorescence in situ hybridization (FISH) (FIG. 1F, FIG. 5c) and quantification of centromere protein foci (FIG. 1G, FIG. 5D; FIG. 6). In addition to their intact karyotype, haploid ES cells did not harbor significant copy number variations (CNVs) relative to their unsorted diploid counterparts (FIG. 5E). Importantly, we did not observe common duplications of specific regions in the two cell lines that would result in pseudo-diploidy. Therefore, genome integrity was preserved throughout haploid-cell isolation and maintenance. As expected, single nucleotide polymorphism (SNP) array analysis demonstrated complete homozygosity of diploid pES10 and pES12 cells across all chromosomes.

Both h-pES10 and h-pES12 exhibited classical human pluripotent stem cell features, including typical colony morphology and alkaline phosphatase activity (FIG. 2A, FIG. 2B). Single haploid ES cells expressed various hallmark pluripotency markers (NANOG, OCT4, SOX2, SSEA4 and TRA1-60), as confirmed in essentially pure haploid cultures by centromere foci quantification (>95% haploids) (FIG. 2C, FIG. 7). Notably, selective flow cytometry enabled to validate the expression of two human ES-cell-specific cell surface markers (TRA-1-60 and CLDN6¹⁸) in single haploid cells (FIG. 2D). Moreover, sorted haploid and diploid ES cells showed highly similar transcriptional and epigenetic signatures of pluripotency genes (FIG. 2E, FIG. 2F). Since the haploid ES cells were derived as parthenotes, they featured distinct transcriptional and epigenetic profiles of maternal imprinting, owing to the absence of paternally-inherited alleles (FIG. 8).

Haploid cells are valuable for loss-of-function genetic screening because phenotypically-selectable mutants can be identified upon disruption of a single allele. To demonstrate the applicability of this principle in haploid human ES cells, we generated a genome-wide mutant library using a piggyBac transposon gene trap system that targets transcriptionally active loci (FIG. 2G, FIG. 8E), and screened for resistance to the purine analog 6-thioguanine (6-TG). Out of six isolated and analyzed 6-TG-resistant colonies, three harbored a gene trap insertion localizing to the nucleoside diphosphate linked moiety X-type motif 5 (NUDT5) autosomal gene (FIG. 2H). NUDT5 disruption was recently confirmed to confer 6-TG resistance in human cells,⁵¹ by acting upstream to the production of 5-phospho-D-ribose-1-pyrophosphate (PRPP), which serves as a phosphoribosyl donor in the hypoxanthine phosphoribosyltransferase 1 (HPRT1)-mediated conversion of 6-TG to thioguanosine monophosphate (TGMP) (FIG. 2I). Detection of a loss-of-function phenotype due to an autosomal mutation validates that genetic screening is feasible in haploid human ES cells.

EXAMPLE 11

Two llamas (No. 146 and No. 147) were immunized with 6 boosts (100 or 50 μg/dose at weekly intervals) of R&D Systems (Minneapolis, MN, US) Cat #1086-PD, which is the ectodomain of human PD1 (rhPD1-Fc), formulated in Titermax Gold (Titermax USA, Norcross, GA, US), according to standard protocols. At week 4, sera were collected to define antibody titers against PD-1 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were coated with rhPD1-Fc. After blocking and adding diluted sera samples, the presence of anti-PD-1 Nanobodies was demonstrated by using rabbit anti-llama immunoglobulin antiserum and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The titer exceeded 16000 for both animals.