Mutation rate in the hypervariable VNTR g3 (D7S22) is affected by allele length and a flanking DNA sequence polymorphism near the repeat array.

The hypervariable human minisatellite locus D7S22 (g3) is highly polymorphic. The allelic distribution in D7S22 features a size clustering of the alleles and a comparably low allelic diversity among small alleles. This reduced diversity could reflect a situation where some alleles are less likely to mutate than others. Several factors could explain such an effect, including allele size, variation in repeat composition, and allelic differences in nearby cis-acting elements affecting the mutation rate. We have characterized 40 de novo mutations found on Southern blots in a large amount of paternity-testing material. There is a significant excess of paternal mutations, and small size changes are most frequent. Mutation rate is affected by allele length, with highest rates in larger alleles. Alleles of the family groups with D7S22 mutations and 50 small alleles were analyzed by nucleotide sequencing. Two hundred thirty-six base pairs of the immediate flanking region upstream of the repeat array were PCR amplified and screened for point mutations by DNA sequencing of the PCR products. Two base substitution polymorphisms were identified: one C/G transversion and one A/G transition, 54 bp and 173 bp upstream of the repeat array, respectively. There is a significant association between mutation and occurrence of 54C, while association is not obvious between mutation rate and the 173A/G variants. There is a marked association between different flanking haplotypes and allele size, and within the smallest allele-size group, all alleles had the 54G/173A haplotype. Both allele size and allelic state at site 54 remain associated with mutation rate when the other factor is controlled. Possible mechanisms behind the variation in mutation rate in D7S22 are discussed.     

Altered Hox expression and increased cell death distinguish Hypodactyly from Hoxa13 null mice.

Hypodactyly (Hoxa13Hd) mice have a small deletion within the coding sequence of Hoxa13 and a limb phenotype that is more severe than that of mice with an engineered null allele of Hoxa13. We used whole-mount in situ hybridization, Nile blue sulfate staining and genetic crosses to determine the basis for the phenotypic differences between these two mutants. Expression of Hoxd13 was unaffected in Hoxa13-/- mice, but its domain was reduced at the anterior and posterior margins of the autopod in Hoxa13Hd/Hd limb buds. The maturation of Hoxd11 expression was delayed and expression of Hoxa11 failed to become restricted to the autopod/zeugopod junction in both Hoxa13Hd/Hd and Hoxa13-/- limb buds compared to wild-type mice. Fgf8 expression was normal in both Hoxa13Hd/Hd and Hoxa13-/- mice throughout limb development. A dramatic increase in cell death was observed in limb bud mesenchyme of Hoxa13Hd/Hd mice as early as E11.5 but not in mice homozygous for the null allele. Genetic background was excluded as the basisforthe phenotypic differences. Compound heterozygotes (Hoxa13-/Hd) displayed an intermediate phenotype relative to both homozygotes suggesting that Hoxa13Hd has an effect on the development of the autopod beyond that which may result from a loss of HOXA13 protein. These results showthat Hoxa13Hd has a negative effect on the survival of the mesenchyme in the autopod, unlike the Hoxa13 null mutation, that cannot be explained by a failure of the AER to express Fgfs. In addition, at least one target of HOXA13 may be Hoxa11.     

Conditional alleles of Msx1 and Msx2

The msh-related homeobox genes, Msx1 and Msx2, have a variety functions during murine organogenesis, Msx1 in the development of the palate and teeth, Msx2 in the skull, teeth, and skin. Msx1 mutants die perinatally. Compound Msx1-2 mutants do not survive past late gestation. The multiplicity of functions of Msx1 and 2, as well as the lethality of Msx1 and Msx1-2 mutants limits the utility of the conventional knockouts. We therefore produced conditional alleles of Msx1 and Msx2. We constructed targeting vectors with LoxP sites flanking the homeodomain-encoding second exons and Frt sites flanking a neo gene. These vectors were used to produce targeted ES cells and mice with floxed alleles. The functionality of the LoxP sites in the floxed alleles was established by crosses with K14-Cre mice (epidermis-specific), and with an Msx2-Cre line that produces a germline deletion. Analysis of progeny by PCR revealed correct Cre-mediated recombination, as well as expected phenotypes.     

A Hoxa13:Cre mouse strain for conditional gene manipulation in developing limb,hindgut, and urogenital system

The developing limb is a useful model for studying organogenesis and developmental processes. Although Cre alleles exist for conditional loss‐ or gain‐of‐function in limbs, Cre alleles targeting specific limb subdomains are desirable. Here we report on the generation of the Hoxa13:Cre line, in which the Cre gene is inserted in the endogenous Hoxa13 gene. We provide evidence that the Cre is active in embryonic tissues/regions where the endogenous Hoxa13 gene is expressed. Our results show that cells expressing Hoxa13 in developing limb buds contribute to the entire autopod (hand/feet) skeleton and validate Hoxa13 as a distal limb marker as far as the skeleton is concerned. In contrast, in the limb musculature, Cre‐based fate mapping shows that almost all muscle masses of the zeugopod (forearm) and part of the triceps contain Hoxa13‐expressing cells and/or their descendants. Besides the limb, the activity of the Cre is detectable in the urogenital system and the hindgut, primarily in the epithelium and smooth muscles. Together our data show that the Hoxa13:Cre allele is a useful tool for conditional gene manipulation in the urogenital system, posterior digestive tract, autopod and part of the limb musculature. genesis 53:366–376, 2015. © 2015 Wiley Periodicals, Inc.     

Generation of a Bmp2 conditional null allele

Bone morphogenetic proteins (Bmp's) are known to play many important roles in embryogenesis. In addition, recent data from human genetic studies has revealed that Bmp's also have important functions in maintenance of the adult phenotype and aging. The original Bmp2 germline null allele resulted in lethality at embryonic day 7.0-10.5 due to malformation of the amnion/chorion and cardiac malformations. Because the early embryonic lethality of the Bmp2 germline null allele hinders further investigation into Bmp2 function at later stages, we generated a Bmp2 conditional null allele. Using gene targeting in mouse embryonic stem (ES) cells, we introduced LoxP sites upstream and downstream of Bmp2 exon 3 that encodes the mature peptide. Our results indicate that the Bmp2 conditional null allele is a true conditional null that encodes wildtype activity and reverts to a null allele after cre recombinase-induced recombination.     

Generation and characterization of a conditional deletion allele for Lmna in mice

Extensive efforts have been devoted to study A-type lamins because mutations in their gene, LMNA in humans, are associated with a number of diseases. The mouse germline mutations in the A-type lamins (encoded by Lmna) exhibit postnatal lethality at either 4–8 postnatal (P) weeks or P16–18 days, depending on the deletion alleles. These mice exhibit defects in several tissues including hearts and skeletal muscles. Despite numerous studies, how the germline mutation of Lmna, which is expressed in many postnatal tissues, affects only selected tissues remains poorly understood. Addressing the tissue specific functions of Lmna requires the generation and careful characterization of conditional Lmna null alleles. Here we report the creation of a conditional Lmna knockout allele in mice by introducing loxP sites flanking the second exon of Lmna. The Lmna^flox/flox mice are phenotypically normal and fertile. We show that Lmna homozygous mutants (Lmna^Δ/Δ) generated by germline Cre expression display postnatal lethality at P16–18 days with defects similar to a recently reported germline Lmna knockout mouse that exhibits the earliest lethality compared to other germline knockout alleles. This conditional knockout mouse strain should serve as an important genetic tool to study the tissue specific roles of Lmna, which would contribute toward the understanding of various human diseases associated with A-type lamins.     

Isoallele Frequencies in Very Large Populations

The frequencies of electrophoretically distinguishable allelic forms of enzymes may be very different from the corresponding frequencies of structurally distinct forms, because many sequence variants may have identical electrophoretic charge. In large populations such frequencies will be determined largely by the number of amino acid sites that are free to vary. The number of distinguishable electrophoretic variants will remain fairly small. Beyond some limiting size, no further effect of population size on allele frequencies is expected, so isolated large populations will have closely similar allele frequencies if polymorphism is due largely to mutation and drift. The most common electrophoretic alleles are expected to be flanked by the next most common, with the rarer alleles increasingly distal. Neither strong selection nor mutation/drift interpretations of enzyme polymorphism are yet disproven, nor is any point between these extremes.     

Mutation discovery in the mouse using genetically guided array capture and resequencing

Forward genetics (phenotype-driven approaches) remain the primary source for allelic variants in the mouse. Unfortunately, the gap between observable phenotype and causative genotype limits the widespread use of spontaneous and induced mouse mutants. As alternatives to traditional positional cloning and mutation detection approaches, sequence capture and next-generation sequencing technologies can be used to rapidly sequence subsets of the genome. Application of these technologies to mutation detection efforts in the mouse has the potential to significantly reduce the time and resources required for mutation identification by abrogating the need for high-resolution genetic mapping, long-range PCR, and sequencing of individual PCR amplimers. As proof of principle, we used array-based sequence capture and pyrosequencing to sequence an allelic series from the classically defined Kit locus (~200 kb) from each of five noncomplementing Kit mutants (one known allele and four unknown alleles) and have successfully identified and validated a nonsynonymous coding mutation for each allele. These data represent the first documentation and validation that these new technologies can be used to efficiently discover causative mutations. Importantly, these data also provide a specific methodological foundation for the development of large-scale mutation detection efforts in the laboratory mouse. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. M. D’Ascenzo and C. Meacham contributed equally to this work.     

Generation of an Fgf9 conditional null allele

The fibroblast growth factor (FGF) family consists of 22 widely expressed regulatory polypeptides and controls a broad spectrum of cellular processes. Accumulating data show that FGF9 plays important roles both in embryogenesis and in adult tissue homeostasis. Ablation of Fgf9 alleles leads to lethality at the neonatal stage mainly due to malformations of the lung, as well as causing male-to-female sex reversal. To circumvent the neonatal lethality resulting from disruption of the Fgf9 gene, which hinders further characterization of the role of FGF9 in adult tissue function and homeostasis, we generated an Fgf9 conditional null allele for spatiotemporal- and tissue-specific disruption of Fgf9. Using gene targeting in mouse embryonic stem (ES) cells, we introduced two loxP sites flanking exon 1 in the Fgf9 allele, which encodes 93 amino acid residues at the N-terminal of FGF9. Our results indicate that the Fgf9 conditional null allele is a true conditional null that encodes wildtype activity and reverts to a null allele after recombination mediated by the Cre recombinase.     

Loss of Eph-receptor expression correlates with loss of cell adhesion and chondrogenic capacity in Hoxa13 mutant limbs.

Mesenchymal patterning is an active process whereby genetic commands coordinate cell adhesion, sorting and condensation, and thereby direct the formation of morphological structures. In mice that lack the Hoxa13 gene, the mesenchymal condensations that form the autopod skeletal elements are poorly resolved, resulting in missing digit, carpal and tarsal elements. In addition, mesenchymal and endothelial cell layers of the umbilical arteries (UAs) are disorganized, resulting in their stenosis and in embryonic death. To further investigate the role of Hoxa13 in these phenotypes, we generated a loss-of-function allele in which the GFP gene was targeted into the Hoxa13 locus. This allele allowed FACS isolation of mesenchymal cells from Hoxa13 heterozygous and mutant homozygous limb buds. Hoxa13(GFP) expressing mesenchymal cells from Hoxa13 mutant homozygous embryos are defective in forming chondrogenic condensations in vitro. Analysis of pro-adhesion molecules in the autopod of Hoxa13 mutants revealed a marked reduction in EphA7 expression in affected digits, as well as in micromass cell cultures prepared from mutant mesenchymal cells. Finally, antibody blocking of the EphA7 extracellular domain severely inhibits the capacity of Hoxa13(GFP) heterozygous cells to condense and form chondrogenic nodules in vitro, which is consistent with the hypothesis that reduction in EphA7 expression affects the capacity of Hoxa13(-/-) mesenchymal cells to form chondrogenic condensations in vivo and in vitro. EphA7 and EphA4 expression were also decreased in the mesenchymal and endothelial cells that form the umbilical arteries in Hoxa13 mutant homozygous embryos. These results suggest that an important role for Hoxa13 during limb and UA development is to regulate genes whose products are required for mesenchymal cell adhesion, sorting and boundary formation.     

EXO1 and MSH6 are high-copy suppressors of conditional mutations in the MSH2 mismatch repair gene of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Msh2p, a central component in mismatch repair, forms a heterodimer with Msh3p to repair small insertion/deletion mismatches and with Msh6p to repair base pair mismatches and single-nucleotide insertion/deletion mismatches. In haploids, a msh2Delta mutation is synthetically lethal with pol3-01, a mutation in the Poldelta proofreading exonuclease. Six conditional alleles of msh2 were identified as those that conferred viability in pol3-01 strains at 26 degrees but not at 35 degrees. DNA sequencing revealed that mutations in several of the msh2(ts) alleles are located in regions with previously unidentified functions. The conditional inviability of two mutants, msh2-L560S pol3-01 and msh2-L910P pol3-01, was suppressed by overexpression of EXO1 and MSH6, respectively. Partial suppression was also observed for the temperature-sensitive mutator phenotype exhibited by msh2-L560S and msh2-L910P strains in the lys2-Bgl reversion assay. High-copy plasmids bearing mutations in the conserved EXO1 nuclease domain were unable to suppress msh2-L560S pol3-01 conditional lethality. These results, in combination with a genetic analysis of msh6Delta pol3-01 and msh3Delta pol3-01 strains, suggest that the activity of the Msh2p-Msh6p heterodimer is important for viability in the presence of the pol3-01 mutation and that Exo1p plays a catalytic role in Msh2p-mediated mismatch repair.     

Temperature sensitive nop2 alleles defective in synthesis of 25S rRNA and large ribosomal subunits in Saccharomyces cerevisiae

Using molecular genetic techniques, we have generated and characterized six temperature sensitive (ts) alleles of nop2. All failed to support growth at 37°C and one was also formamide sensitive (fs) and failed to grow on media containing 3% formamide. Conditional lethality is not due to rapid turnover of mutant Nop2p proteins at 37°C. Each allele contains between seven and 14 amino acid substitutions and one possesses a nonsense mutation near the C-terminus. Mapping experiments with one allele, nop2-4, revealed that a subset of the amino acid substitutions conferred the ts phenotype and that these mutations have an additive effect. All six mutants exhibited dramatic reductions in levels of 60S ribosome subunits under non-permissive conditions as well as some reduction at permissive temperature. Processing of 27S pre-rRNA to mature 25S rRNA was defective in all six mutants grown under non-permissive conditions. Levels of the 40S ribosomal subunit and 18S rRNA were not significantly affected. Amino acid substitutions in nop2 conditional alleles are discussed in the context of the hypothesis that Nop2p functions both as an RNA methyltransferase and a trans-acting factor in rRNA processing and large ribosomal subunit biogenesis.     

Spontaneous Allozyme Mutations in DROSOPHILA MELANOGASTER: Rate of Occurrence and Nature of the Mutants

After additional generations of accumulation of allozyme mutants, the 1,000 lines of Mukai and Cockerham (1977) were again screened for the same five loci (alpha-Gpdh, cMdh, Adh, Hex-C and alpha-Amy), as well as for two new loci (Got-2 and Dip-A). Based on 3,111,598 allele generations: (1) the average mutation rate to new mobility variants with normal function was estimated to be 1.28 x 10(-6), and (2) the average mutation rate to null alleles was estimated to be 3.86 x 10(-6). A qualitative analysis of the nulls provided evidence that most of the mutants recovered are due to base substitutions. No apparent correlation was observed between structural gene size and mutation rate.     

Generation of novel conditional and hypomorphic alleles of the Smad2 gene

Smad2 is an intracellular mediator of the transforming growth factor beta signaling (TGFbeta) pathway. It has been previously shown that, in the mouse, ablation of functional Smad2 results in embryonic lethality due to gastrulation defects. To circumvent the early lethality and study the spatially and temporally specific functions of Smad2, we utilized the Cre-loxP system to generate a Smad2 conditional allele. Here we show that a conditional allele, Smad2(flox), was generated. In this allele, exons 9 and 10 are flanked by loxP sites and the gene is functionally wildtype. Cre-mediated recombination results in a deletion allele which phenocopies our previously reported Smad2(DeltaC) null mutation. To generate this conditional allele, we first made a targeted mutation which introduced a floxed neo cassette into intron 10. This allele (Smad2(3loxP)) functions hypomorphically when placed opposite a null allele, and unlike the other published Smad2 hypomorphic allele, can be maintained in the homozygous state.