Genetic mapping of species differences via in vitro crosses in mouse embryonic stem cells

Lazzarano, Stefano; Kučka, Marek; Castro, João Pl; Naumann, Ronald; Medina, Paloma; Fletcher, Michael; Wombacher, Rebecka; Gribnau, Joost; Hochepied, Tino; Montagu, Marc Van; Libert, Claude; Chan, Yingguang Frank

doi:10.1073/pnas.1717474115

Cited by 10 publications

(7 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This translates to an endogenous rate of ∼0.8 recombination events per cell per generation and an increased rate of approximately one recombination event per cell per generation after ML216 treatment. These apparent rates in allotetraploid cells are substantially higher than previously reported mitotic recombination rates in diploid human and mouse embryonic stem cells [0.01 to 0.04 recombination events per cell per generation after ML216 treatment; SI Appendix, Supplemental Materials and Methods (10,11,29)]. Further investigation will be required to assess whether ML216 significantly increases the rate of interspecific recombination and whether other small molecules such as camptothecin can also increase the rate of recombination events between human and chimpanzee chromosomes in allotetraploid iPSCs.…”

Section: Differential Gene Expression and Allele-specific Gene Expressionmentioning

confidence: 66%

“…induced by Cas9 and guide RNAs (gRNAs) designed to alter particular locations in the genome. Mutations and chemical inhibitors of the Bloom Syndrome helicase gene (BLM) have been used to recover homozygous mutants in somatic cell gene screens (9,10) or to induce recombination between chromosomes of distantly related mouse strains for studies of the genomic basis of evolutionary differences (11). The ability to induce breaks at particular loci with CRISPR-Cas9 has also made it possible to choose both the location and the direction of recombination between genomes in nonmeiotic cells, enabling high-resolution mapping without traditional crosses in yeast (12).…”

Section: Significancementioning

confidence: 99%

“…Further localization of both cisand trans-regulatory differences would be greatly aided if it were possible to generate mapping panels that carry different dosages of human and chimpanzee alleles at known locations throughout the genome. Previous studies in yeast, Drosophila, and cultured mammalian cell lines have used mitotic recombination to generate useful mapping panels from somatic cells (11,12,21,22). To boost the rate of mitotic recombination, common strategies have been to treat cells with small molecules that promote DNA damage (23,24), or to induce targeted recombination at specific loci using CRISPR-Cas9 (12,21).…”

Section: Differential Gene Expression and Allele-specific Gene Expressionmentioning

confidence: 99%

See 2 more Smart Citations

Genetic studies of human–chimpanzee divergence using stem cell fusions

Song

Grant

Behrens

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Complete genome sequencing has identified millions of DNA changes that differ between humans and chimpanzees. Although a subset of these changes likely underlies important phenotypic differences between humans and chimpanzees, it is currently difficult to distinguish causal from incidental changes and to map specific phenotypes to particular genome locations. To facilitate further genetic study of human–chimpanzee divergence, we have generated human and chimpanzee autotetraploids and allotetraploids by fusing induced pluripotent stem cells (iPSCs) of each species. The resulting tetraploid iPSCs can be stably maintained and retain the ability to differentiate along ectoderm, mesoderm, and endoderm lineages. RNA sequencing identifies thousands of genes whose expression differs between humans and chimpanzees when assessed in single-species diploid or autotetraploid iPSCs. Analysis of gene expression patterns in interspecific allotetraploid iPSCs shows that human–chimpanzee expression differences arise from substantial contributions of both cis-acting changes linked to the genes themselves and trans-acting changes elsewhere in the genome. To enable further genetic mapping of species differences, we tested chemical treatments for stimulating genome-wide mitotic recombination between human and chimpanzee chromosomes, and CRISPR methods for inducing species-specific changes on particular chromosomes in allotetraploid cells. We successfully generated derivative cells with nested deletions or interspecific recombination on the X chromosome. These studies confirm an important role for the X chromosome in trans regulation of expression differences between species and illustrate the potential of this system for more detailed cis and trans mapping of the molecular basis of human and chimpanzee evolution.

show abstract

Section: Differential Gene Expression and Allele-specific Gene Expressionmentioning

confidence: 66%

Section: Significancementioning

confidence: 99%

Section: Differential Gene Expression and Allele-specific Gene Expressionmentioning

confidence: 99%

See 1 more Smart Citation

Genetic studies of human–chimpanzee divergence using stem cell fusions

Song

Grant

Behrens

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…More recently, Lazzarano et al showed that ML216 induces mitotic crossover in mouse ESCs. They also reported that transient suppression of the BLM protein did not increase aneuploidy [13]. The procedure used to isolate positive clones generated with the BLM inhibitor ML216 is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Generation of targeted homozygosity in the genome of human induced pluripotent stem cells

et al. 2019

View full text Add to dashboard Cite

When loss of heterozygosity (LOH) is correlated with loss or gain of a disease phenotype, it is often necessary to identify which gene or genes are involved. Here, we developed a region-specific LOH-inducing system based on mitotic crossover in human induced pluripotent stem cells (hiPSCs). We first tested our system on chromosome 19. To detect homozygous clones generated by LOH, a positive selection cassette was inserted at the AASV1 locus of chromosome 19. LOHs were generated by the combination of allele-specific double-stranded DNA breaks introduced by CRISPR/Cas9 and suppression of Bloom syndrome (BLM) gene expression by the Tet-Off system. The BLM protein inhibitor ML216 exhibited a similar crossover efficiency and distribution of crossover sites. We next applied this system to the short arm of chromosome 6, where human leukocyte antigen (HLA) loci are located. Genotyping and flow cytometric analysis demonstrated that LOHs associated with chromosomal crossover occurred at the expected positions. Although careful examination of HLA-homozygous hiPSCs generated from parental cells is needed for cancer predisposition and effectiveness of differentiation, they may help to mitigate the current shortcoming of hiPSC-based transplantation related to the immunological differences between the donor and host.

show abstract

“…To date, the field has advanced largely on the strength of candidate-based studies that implicate a single variant gene in an interspecific trait 5,6 , with the complete genetic architecture often remaining unknown. Against the backdrop of a few specialized introgression [7][8][9][10] and molecular-evolution 11 techniques available in the field, dissection of complex trait differences between species has remained a key challenge.…”

mentioning

confidence: 99%