Generation of conditional knockout alleles for PDGF-C

PDGF-C is a newly identified member of the platelet-derived growth factor (PDGF) family, which is involved in multiple cellular functions by signaling through PDGF receptor (PDGFR)-alphaalpha and alphabeta dimers. PDGF-C deficiency is perinatal lethal due to the formation of cleft palate. To further characterize the cellular function of PDGF-C during both embryonic and postnatal development, we have generated two conditional alleles of the Pdgf-c gene in which two loxP sites flank exon 5. Global Cre-mediated excision of the floxed exon 5 in these alleles resulted in a complete loss of PDGF-C expression and caused embryonic defects identical to those previously described for the PDGF-C null embryos. These conditional alleles will therefore be the important genetic tools for dissecting the spatial and temporal roles of PDGF-C during development and in adult tissues. Furthermore, from this work, we have also described a simple approach for creating mouse conditional alleles in an efficient manner.     

Bi-FoRe:an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function.However,contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported.Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system.We demonstrated the feasibility of this strategy at sox10 and isl1 loci,and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter,allowing generation of genetic mosaics for lineage tracing.We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles,both tagged with two different fluorescent reporters.By introducing Cre recombinase,these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel;furthermore,differential fluorescent labeling of the positive and negative alleles enables simple,early and efficient realtime discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes.We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus.Furthermore,we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology.Our system could easily be expanded for other applications or to other organisms,and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.     

High‐throughput identification of conditional MHCI ligands and scaled‐up production of conditional MHCI complexes

Despite the need to monitor the impact of Cancer Immunotherapy (CI)/Immuno‐Oncology (IO) therapeutics on neoantigen‐specific T‐cell responses, very few clinical programs incorporate this aspect of immune monitoring due to the challenges in high‐throughput (HTP) generation of Major Histocompatibility Complex Class I (MHCI) tetramers across a wide range of HLA alleles. This limitation was recently addressed through the development of MHCI complexes with peptides containing a nonnatural UV cleavable amino acid (conditional MHCI ligands) that enabled HTP peptide exchange upon UV exposure. Despite this advancement, the number of alleles with known conditional MHCI ligands is limited. We developed a novel workflow to enable identification and validation of conditional MHCI ligands across a range of HLA alleles. First, known peptide binders were screened via an enzyme‐linked immunosorbent assay (ELISA) assay. Conditional MHCI ligands were designed using the highest‐performing peptides and evaluated in the same ELISA assay. The top performers were then selected for scale‐up production. Next‐generation analytical techniques (LC/MS, SEC‐MALS, and 2D LC/MS) were used to characterize the complex after refolding with the conditional MHCI ligands. Finally, we used 2D LC/MS to evaluate peptide exchange with these scaled‐up conditional MHCI complexes after UV exposure with validated peptide binders. Successful peptide exchange was observed for all conditional MHCI ligands upon UV exposure, validating our screening approach. This approach has the potential to be broadly applied and enable HTP generation of MHCI monomers and tetramers across a wider range of HLA alleles, which could be critical to enabling the use of MHCI tetramers to monitor neoantigen‐specific T‐cells in the clinic.     

On the Divergence of Alleles in Nested Subsamples from Finite Populations

Within-population variation at the DNA level will rarely be studied by sequencing of loci of randomly chosen individuals. Instead, individuals will usually be chosen for sequencing based on some knowledge of their genotype. Data collected in this way require new sampling theory. Motivated by these observations, we have examined the sampling properties of a finite population model with two mutation processes and with no selection or recombination. One mutation process generates new alleles according to an infinite-alleles model, and the other generates polymorphisms at sites according to an infinite-sites model. A sample of n genes is considered. The stationary distribution of the number of segregating sites in a subsample from one of the allelic classes in the sample conditional on the allelic configuration of the sample is studied. A recursive scheme is developed to compute the moments of this distribution, and it is shown that the distribution is functionally independent of the number of additional alleles in the sample and their respective frequencies in the sample. For the case in which the sample contains only two alleles, the distribution of the number of segregating sites in a subsample containing both alleles conditional on the sample frequencies of the alleles is studied. The results are applied to the analysis of DNA sequences of two alleles found at the Adh locus of Drosophila melanogaster. No significant departure from the neutral model is detected.     

High throughput gene trapping and postinsertional modifications of gene trap alleles

Gene trapping is a high-throughput insertional mutagenesis approach that has been primarily used in mouse embryonic stem cells (ESCs). As a high throughput technology, gene trapping helped to generate tenth of thousands of ESC lines harboring mutations in single genes that can be used for making knock-out mice. Ongoing international efforts operating under the umbrella of the International Knockout Mouse Consortium (IKMC; www.knockoutmouse.org) aim to generate conditional alleles for every protein coding gene in the mouse genome by high throughput conditional gene targeting and trapping. Here, we provide protocols for gene trapping in ESCs that can be easily adapted to any other mammalian cell. We further provide protocols for handling and verifying conditional gene trap alleles in ESC lines obtained from the IKMC repositories and describe a highly efficient method for the postinsertional modification of gene trap alleles. More specifically, we describe a protein tagging strategy based on recombinase mediated cassette exchange (RMCE) that enables protein localization and protein-protein interaction studies under physiological conditions.     

Derivation of single-locus relationship coefficients conditional on marker information

The coefficient of relationship is defined as the correlation between the additive genetic values of two individuals. This coefficient can be defined specifically for a single quantitative trait locus (QTL) and may deviate considerably from the overall expectation if it is taken conditional on information from linked marker loci. Conditional halfsib correlations are derived under a simple genetic model with a biallelic QTL linked to a biallelic marker locus. The conditional relationship coefficients are shown to depend on the recombination rate between the marker and the QTL and the population frequency of the marker alleles, but not on parameters of the QTL, i.e. number and frequency of QTL alleles, degree of dominance etc., nor on the (usually unknown) QTL genotype of the sire. Extensions to less simplified cases (multiple alleles at the marker locus and the QTL, two marker loci flanking the QTL) are given. For arbitrary pedigrees, conditional relationship coefficients can also be derived from the conditional gametic covariance matrix suggested by Fernando and Grossman (1989). The connection of these two approaches is discussed. The conditional relationship coefficient can be used for marker-assisted genetic evaluation as well as for the detection of QTL and the estimation of their effects.     

Insertional Mutations in the Yeast Hop1 Gene: Evidence for Multimeric Assembly in Meiosis

The HOP1 gene of Saccharomyces cerevisiae has been shown to play an important role in meiotic synapsis. In this study we analyzed the mechanism of this function by phenotypic characterization of novel in-frame linker-insertion mutations located at various sites throughout the HOP1 coding sequence. Among 12 mutations found to cause defects in meiotic recombination and spore viability, three were temperature-sensitive for the spore viability defect. Although substantial meiotic recombination was found for these conditional alleles at the restrictive temperature, the level of exchange measured in spo13 meiosis was reduced in some of the monitored intervals, indicating that nondisjunction resulting from a deficit in crossing over could account for SPO13 spore inviability. Intragenic complementation between linker-insertion alleles was assessed by testing the viability of spores generated from heteroallelic diploids after SPO13 meiosis. Complex patterns of complementation and enhancement of the spore-inviability phenotype indicate that HOP1 functions in a multimeric complex. In addition, the ability of alleles which map near the carboxyl terminus to complement several other alleles provides evidence for a functional domain in this region of the protein. Two previously identified multicopy suppressors of the conditional hop1-628(ts) allele were tested for their effects in cells bearing the linker-insertion hop1 alleles. Overexpression of REC104 from a 2μ plasmid was shown to enhance the spore viability of every allele tested, including a hop1 disruption allele. On the other hand, suppression by overexpression of RED1 from a 2μ plasmid was found only for allele hop1-628(ts). Surprisingly, similar overexpression of RED1 in strains bearing several other conditional hop1 linker-insertion alleles caused enhanced spore lethality. This finding, in conjunction with the evidence for a carboxy-terminal domain, provides new insight into the nature of interactions between the HOP1 and RED1 products in meiosis.     

Generation of a set of conditional analog-sensitive alleles of essential protein kinases in the fission yeast Schizosaccharomyces pombe

The genome of the fission yeast Schizosaccharomyces pombe encodes for 17 protein kinases that are essential for viability. Studies of the essential kinases often require the use of mutant strains carrying conditional alleles. To inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. The mutation of a single residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. Using this approach, we constructed conditional analog-sensitive alleles of 13 essential protein kinases in the fission yeast S. pombe.Key words: kinase, analog-sensitive, conditional allele, fission yeast, phosphorylation     

Characterization of non-dominant lethal mutations in the yeast plasma membrane H+-ATPase gene

Site-directed mutants of yeast ATPase were previously studied after introduction of mutant alleles into a yeast strain where these alleles were constitutively expressed while the expression of the wild-type chromosomal ATPase gene was turned off. As a functional H+ pump is essential, strong selective pressure leads to the accumulation of revertants during growth of cells harboring variants with low activity. Thus, constitutive expression of the mutant gene can select phenotypes which reflect events such as gene conversion or reversion. We have therefore re-evaluated the phenotypes of non-dominant lethal alleles in an alternative set of conditional expression systems. We show that eight of 11 previously described site-directed mutations behave as recessive lethal alleles.     

Generation of a set of conditional analog-sensitive alleles of essential protein kinases in the fission yeast Schizosaccharomyces pombe

The genome of the fission yeast Schizosaccharomyces pombe encodes for 17 protein kinases that are essential for viability. Studies of the essential kinases often require the use of mutant strains carrying conditional alleles. To inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. The mutation of a single residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. Using this approach, we constructed conditional analog-sensitive alleles of 13 essential protein kinases in the fission yeast S. pombe.     

Balancer-Cre transgenic mouse germ cells direct the incomplete resolution of a tri-loxP-targeted Cyp1a1 allele, producing a conditional knockout allele

To generate conditional alleles, genes are commonly engineered to contain recognition sites for bacteriophage recombinases, such as Cre recombinase. When such motifs (lox sites) flank essential gene sequences, and provided that Cre recombinase is expressed, Cre recombinase will excise the flanked sequence-creating a conditional knockout allele. Targeted conditional alleles contain a minimum of three lox sites. It would be desirable to have Cre recombinase perform partial resolution (i.e., recombination some of the time between only the two lox sites flanking the marker gene). Here we report use of the commercially available Balancer2-Cre transgenic mouse line to carry out this function from a tri-loxP-site-containing cytochrome p450 1A1 (Cyp1a1) targeted allele. Such incomplete resolution of this complex locus occurred progressively with age in germ cells of male mice; the conditional Cyp1a1 gene was recovered in offspring from mice containing the targeted Cyp1a1 allele and the Cre recombinase transgene. Removal of the marker gene resulted in a conditional Cyp1a1 allele whose expression was indistinguishable from that of the wild-type allele.     

Bi-allelic gene targeting in mouse embryonic stem cells

The EUCOMM and KOMP programs have generated targeted conditional alleles in mouse embryonic stem cells for nearly 10,000 genes. The availability of these stem cell resources will greatly accelerate the functional analysis of genes in mice and in cultured cells. We present a method for conditional ablation of genes in ES cells using vectors and targeted clones from the EUCOMM and KOMP conditional resources. Inducible homozygous cells described here provide a precisely controlled experimental system to study gene function in a model cell.     

Conditional allelic replacement applied to genes encoding the histone variant H3.3 in the mouse

Post‐translational modifications to residues in core histones convey epigenetic information. Their function can be evaluated in amino acid substitution mutants, although to date this method has not been used in mice. To this end, we have evaluated gene targeting vectors designed for Cre recombinase‐mediated conditional allelic replacement at the two unlinked genes encoding the histone variant H3.3. The conditional alleles consist of an uninterrupted wild‐type H3.3 coding sequence upstream of a desired alternative or proxy coding sequence. The arrangement of two loxP sites allows Cre‐mediated replacement of the wild‐type coding sequence with the proxy. To demonstrate proof of principle, at each locus we replaced the wild‐type coding sequence with a fluorescent reporter. This produced null alleles that will be useful to analyse the effects of H3.3 deficiency in development. Each targeting vector can readily be retrofitted with a proxy coding sequence encoding a modified H3.3 protein. Such vectors will allow for the conditional substitution of specific residues in order to dissect the roles of H3.3 post‐translational modifications in development and disease. genesis, 51:142?146, 2013. © 2013 Wiley Periodicals, Inc.     

Tackling an essential problem in functional proteomics of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Gene inactivation is the cornerstone of functional genetic analysis, but the analysis of essential genes requires conditional inactivation of the gene product. A new study has adapted a simple method for creating conditional alleles to allow large-scale analysis of essential genes in Saccharomyces cerevisiae and has identified a role in DNA replication for a newly identified protein complex.     

The conditional ancestral selection graph with strong balancing selection

Using a heuristic separation-of-time-scales argument, we describe the behavior of the conditional ancestral selection graph with very strong balancing selection between a pair of alleles. In the limit as the strength of selection tends to infinity, we find that the ancestral process converges to a neutral structured coalescent, with two subpopulations representing the two alleles and mutation playing the role of migration. This agrees with a previous result of Kaplan et al., obtained using a different approach. We present the results of computer simulations to support our heuristic mathematical results. We also present a more rigorous demonstration that the neutral conditional ancestral process converges to the Kingman coalescent in the limit as the mutation rate tends to infinity.     

Allele excess at neutrally evolving microsatellites and the implications for tests of neutrality

Skews in the observed allele-frequency spectrum are frequently viewed as an indication of non-neutral evolution. Recent surveys of microsatellite variability have used an excess of alleles as a statistical approach to infer positive selection. Using neutral coalescent simulations we demonstrate that the mean numbers of alleles expected under the stepwise-mutation model and infinite-allele model deviate from the observed numbers of alleles. The magnitude of this difference is dependent on the sample size, mutation rates (theta-values) and observed gene diversities. Moreover, we show that the number of observed alleles differs among loci with the same observed gene diversity but different mutation rates (theta-values). We propose that a reliable test statistic based on allele excess must determine the confidence interval by computer simulations conditional on the observed gene diversity and theta-values. As the latter are notoriously difficult to obtain for experimental data, we suggest that other statistics, such as lnRV, may be better suited to the identification of microsatellite loci subject to selection.