Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection.

CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies.

Quantitative methods for studying hemostasis in zebrafish larvae.

Hemostasis is a coordinated system through which blood is prevented from exiting a closed circulatory system. We have taken advantage of the zebrafish, an emerging model for the study of blood coagulation, and describe three techniques for quantitative analysis of primary and secondary hemostasis. Collectively, these three techniques comprise a toolset to aid in our understanding of hemostasis and pathological clotting.

Genetic regulation of plasma von Willebrand factor levels: quantitative trait loci analysis in a mouse model.

BACKGROUND: The genetic factors responsible for the wide variation in plasma von Willebrand factor (VWF) levels observed among individuals are largely unknown, although these genes are also likely to contribute to variability in the severity of von Willebrand disease (VWD) and other bleeding and thrombotic disorders. We have previously mapped two genes contributing to the regulation of plasma VWF levels in mice (Mvwf1 on chromosome 11 and Mvwf2 on chromosome 6). OBJECTIVE: To identify additional quantitative trait loci (QTL) contributing to the genetic regulation of murine plasma VWF levels. METHODS: To map genetic loci contributing to the > 7-fold difference in plasma VWF levels between two mouse strains (A/J and CASA/RkJ), high-density individual genotyping and R/qtl analyses were applied to a previously generated set of approximately 200 F2 mice obtained from an intercross of these two inbred lines. RESULTS: Genomic loci for two additional candidate VWF modifier genes were identified: Mvwf3 on chromosome 4 and Mvwf4 on chromosome 13. These loci demonstrate primarily epistatic effects when co-inherited with two CASA/RkJ Vwf alleles, although Mvwf4 may also exert a small, independent, additive effect. CONCLUSIONS: Mvwf3 and Mvwf4, combined with the effect of Mvwf2, explain approximately 45% of the genetic variation in plasma VWF level among the A/J and CASA/RkJ strains. Mvwf3 and Mvwf4 exhibit homology of synteny to three human chromosomal segments (on chromosomes 1, 5 and 6) previously reported by the Genetic Analysis of Idiopathic Thrombophilia (GAIT) study, suggesting that orthologs of Mvwf3 and Mvwf4 may also encode important VWF modifier genes in humans.

Use of the pinna reflex as a test of hearing in mutant mice.

Although it is a gross measure, the pinna reflex test is easily administered and is, therefore, incorporated as a general screening tool in mutagenesis programs. Our recent application of this approach indicated that mutant mice lacking one of the small Maf proteins, in this case MafG, failed to exhibit a pinna reflex. In contrast, littermate controls, with the same mixed 129/CD1 background, and including both wild type and heterozygous mutant animals, passed the test. Because previous studies indicate that mafG is expressed in both cochlear and vestibular parts of the mouse inner ear, the source of this 'presumed deafness' was further assessed by making round window recordings to determine compound action potential thresholds. Auditory brainstem responses were also acquired to assess function along portions of the central auditory pathway. In all cases, responses in homozygous mutants (-/-) were comparable to those obtained from littermate controls, either wild type (+/+) or heterozygous mutants (+/-). Gross anatomy of the organ of Corti was also found to be similar in all three groups of mice. Hence, the lack of a pinna reflex must relate to nonauditory causes.

Perinatal synthetic lethality and hematopoietic defects in compound mafG::mafK mutant mice.

Prior studies exploring the mechanisms controlling erythroid gene regulation implicated MARE (Maf recognition element) cis-elements as crucial to the transcriptional activity of many erythroid genes. Numerous transcription factors can elicit responses through MAREs, including not only the AP-1 family proteins, but also a growing list of factors composed of Cap-N-Collar (CNC)-small Maf heterodimers. While these factors can activate transcription from MAREs in co-transfection assays, mouse germline mutations in cnc genes tested to date have failed to reveal primary erythroid phenotypes. Here we report that after combining the mafK and mafG targeted null alleles, mutant animals display several synthetic phenotypes, including erythroid deficiencies. First, compound homozygous small maf gene mutants survive embryogenesis, but die postnatally. Secondly, compound mutant animals develop severe neurological disorders. Thirdly, they exhibit an exacerbated mafG deficiency in megakaryopoiesis, specifically in proplatelet formation, resulting in profound thrombocytopenia. Finally, the compound mutant animals develop severe anemia accompanied by abnormal erythrocyte morphology and membrane protein composition. These data provide direct evidence that the small Maf transcription factors play an important regulatory role in erythropoiesis.

Positive or negative MARE-dependent transcriptional regulation is determined by the abundance of small Maf proteins.

The small Maf transcription factor proteins bind to Maf Recognition Elements (MAREs) by dimerizing with CNC proteins or themselves. We undertook experiments to clarify the functional relationship between the small Mafs and their partners in vivo. Embryos expressing abundant transgene-derived MafK died of severe anemia, while lines expressing lower levels of small Maf lived to adulthood. Megakaryocytes from the latter overexpressing lines exhibited reduced proplatelet formation and MARE-dependent transcription, phenocopying mafG null mutant mice. When the mafG null mutants were bred to small Maf-overexpressing transgenic animals, both loss- and gain-of-function phenotypes were reversed. These results provide direct in vivo evidence that transcriptional regulation through MARE elements hinges on an exquisitely sensitive balance of activating CNC molecules and their small Maf partners.

Characterization of the murine mafF gene.

Small Maf proteins are obligatory heterodimeric partner molecules of mammalian Cap'n'Collar proteins that together control a wide variety of eukaryotic genes. Although both MafK and MafG are expressed in overlapping but distinct tissue distribution patterns during embryonic development, the physiological consequences of loss-of-function mutations in either gene are modest. This suggested that compensation by the third small Maf protein, MafF, might be a major reason for such mild phenotypes and that further analysis of MafF might therefore provide important insights for understanding small Maf regulatory function(s). We therefore cloned, mapped, transcriptionally and developmentally characterized, and finally disrupted the mafF gene. We show that murine mafF is transcriptionally regulated by three different promoters and is most abundantly expressed in the lung. The lacZ gene inserted into the mafF locus revealed prominent expression sites in the gut, lung, liver, outflow tract of the heart, cartilage, bone membrane, and skin but not in hematopoietic cells at any developmental stage. Homozygous mafF null mutant mice were born in a normal Mendelian ratio and displayed no obvious functional deficiencies, indicating that MafF activity may be dispensable even in tissues where the expression of other small Maf proteins is quite low.

Impaired megakaryopoiesis and behavioral defects in mafG-null mutant mice.

The small Maf proteins (MafG, MafK, and MafF), which serve as heterodimeric partner molecules of CNC family proteins for binding in vitro to MARE sites, have been implicated in the regulation of both transcription and chromatin structure, but there is no current evidence that the proteins fulfill these functions in vivo. To elucidate possible contributions of the small Maf proteins to gene regulation, we have ablated the mafG and mafK genes in mice by replacing their entire coding sequences with the Escherichia coli lacZ gene. mafG homozygous mutant animals exhibit impaired platelet formation accompanied by megakaryocyte proliferation, as well as behavioral abnormalities, whereas mafK-null mutant mice are phenotypically normal. Characterization of the mafG and mafK embryonic expression patterns show that their developmental programs are distinct and intersecting, but not entirely overlapping. These results provide direct evidence that the small Maf transcription factors are vital participants in embryonic development and cellular differentiation.

The world according to Maf.

Maf family proteins are so named because of their structural similarity to the founding member, the oncoprotein v-Maf. The small Maf proteins (MafF, MafG and MafK), as do all family members, include a characteristic basic region linked to a leucine zipper (b-Zip) domain which mediate DNA binding and subunit dimerization respectively. The small Maf proteins form homodimers or heterodimers with other b-Zip proteins present in the cell and bind to Maf recognition elements (MARE) in DNA. Since they lack known transcriptional activation domains, the small Maf proteins function either as obligatory heterodimeric partner molecules with numerous large subunits, discussed below, or alternatively as homo- or heterodimeric transcriptional repressors. The three small Maf proteins are expressed in a number of overlapping tissues, but their expression profiles nonetheless appear to be under meticulous tissue- and developmental stage-specific control. The MARE bears a striking resemblance to the NF-E2 binding sequence. NF-E2 binding sites in the human beta-globin locus control region have been directly implicated as integral components in the circuitry required for eliciting changes in chromatin structure that precede globin gene activation. While the NF-E2 DNA sequence has been shown to be important for erythroid-specific gene regulation, a growing list of other genes may also be regulated through the same, or very similar, cis elements in non-erythroid cells. Taken together, these observations argue that comprehensive analysis of the activities of the small Maf proteins may provide a unique perspective for expanding our understanding of transcriptional regulation that can be elicited through interacting transcription factor networks.

Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4.

Proper tissue- and developmental stage-specific transcriptional control over the five genes of the human beta-globin locus is elicited in part by the locus control region (LCR), but the molecular mechanisms that dictate this determined pattern of gene expression during human development are still controversial. By use of homologous recombination in yeast to generate mutations in the LCR within a yeast artificial chromosome (YAC) bearing the entire human beta-globin gene locus, followed by injection of each of the mutated YACs into murine ova, we addressed the function of LCR hypersensitive site (HS) elements 3 and 4 in human beta-globin gene switching. The experiments revealed a number of unexpected properties that are directly attributable to LCR function. First, deletion of either HS3 or HS4 core elements from an otherwise intact YAC results in catastrophic disruption of globin gene expression at all erythroid developmental stages, despite the presence of all other HS elements in the YAC transgenes. If HS3 is used to replace HS4, gene expression is normal at all developmental stages. Conversely, insertion of the HS4 element in place of HS3 results in significant expression changes at every developmental stage, indicating that individual LCR HS elements play distinct roles in stage-specific beta-type globin gene activation. Although the HS4 duplication leads to alteration in the levels of epsilon- and gamma-globin mRNAs during embryonic erythropoiesis, total beta-type globin mRNA synthesis is balanced, thereby leading to the conclusion that all of the human beta-locus genes are competitively regulated. In summary, the human beta-globin HS elements appear to form a single, synergistic functional entity called the LCR, and HS3 and HS4 appear to be individually indispensable to the integrity of this macromolecular complex.