About Haplobank

Some organisms such as yeast are haploid, i.e. they carry a single set of chromosomes, and thus provide a basis for genetic analyses where recessive mutations of genes will show a clear phenotype due to the absence of a second gene copy. However, all somatic mammalian cells carry two copies of chromosomes (diploidy) that complicate mutational screens. In 2011, while still a postdoc in Josef Penninger"s lab, Uli Elling described the successful generation of mammalian haploid embryonic stem cells from parthenogenetic mouse embryos (Elling et al. (2011), Cell Stem Cell, 9(6):563-74). Haploid mESCs show stable growth over multiple passages, can be efficiently subcloned, and differentiate in vitro and in vivo. Importantly, haploid mESCs can be readily mutagenized at the haploid state to generate complete, homozygous mutants for virtually any gene allowing for reverse and high-throughput forward genetic screens.

 

To account for optimal genomic coverage, we have developed and optimized various conditional transposon-, lentiviral-, and retroviral-based mutagenic gene trap vectors (see below). Each introduced single mutation carries a unique internal DNA barcode, which can be used for rapid validation of mutations as well as for quantitative readouts for the presence and absence of particular mutations, i.e. for identifying depleting mutations.

Using these optimized mutagenesis systems, we launched Haplobank, a repository of homozygously targeted ES cell lines for functional genomics. In order to allow for such a high-throughput, we streamlined and automatized cell culture, DNA preparation, and deep sequencing protocols using a Hamilton robotic platform. The Haplobank team picked a total of 124’128 clones and processed them for integration site analyses. Our optimized 4D pooling/iPCR/NGS approach allows us to retrieve the information on genomic integration sites in about 81% of clones, which translates to 100"351 fully annotated clones. We hit a combined total of 16"970 genes when we count clones with either single (clones are shown as "blue clones" in the browser) or multiple gene trap insertions ("grey clones", intragenic: Intron, ncExon, 1Intron, 5"UTR, CDS). Taking only those clones, which carry a single gene trap, we hit 11"703 unique genes. The current status is summarized below:

Since its start 2013 the Haplobank Team has already expanded and distributed over 1000 clones from the collection for such diverse projects as e.g. cardiomyocyte differentiation or sprouting angiogenesis. These new technologies open the possibility to combine the power of a haploid genome with pluripotency of embryonic stem cells to uncover fundamental developmental and biological processes in defined cell types at the genomic level.

 

Haplobank is in part financed and supported by Nestle and Novartis.