FVB/N (H2 q ) mouse is resistant to arthritis induction and exhibits a genomic deletion of T-cell receptor V beta gene segments

 Animal models of autoimmune diseases have been instrumental in advancing our understanding of autoimmunity in humans. Collagen-induced arthritis (CIA) in mice is an autoimmune disease model of rheumatoid arthritis. Susceptibility to CIA in mice is linked to genes of the major histocompatibility complex (MHC). CD4⁺ T cells that express the T-cell receptor (TCR) Tcra-V11.1 and/or Tcrb-V8.2 play a key role in the pathogenesis of arthritis in the DBA/1 mouse (H2 ^q ). We identified an inbred mouse strain, FVB/NJ (H2 ^q ), that is resistant to arthritis induction and exhibits a genomic deletion of certain Tcrb-V gene segments. We report a novel polymerase chain reaction-based method for the rapid identification of new mouse strains that exhibit germline Tcrb-V gene deletions. We mapped for the first time both the 5′ and 3′ breakpoints of the Tcrb-V deletion in the FVB/NJ, SWR, SJL, C57L, and C57BR strains to within 1.1 kilobases. Since there is an association between a particular Tcra-V allele (Tcra-V11.1 ^d ) and arthritis susceptibility in H2 ^q mouse strains, we examined the allelic polymorphisms of the Tcra-V11 gene subfamily members between the arthritis-susceptible DBA/1 mouse and the arthritis-resistant FVB/NJ mouse strain. The amino acid sequences of the Tcra-V11.1 alleles differ at two positions (codons 18 and 68). Therefore, the resistance of FVB/NJ mouse to arthritis induction may be due in part to Tcra-V11.1 coding sequence polymorphism and Tcrb-V8.2 gene segment deletion, as we have recently demonstrated in the case of SWR mouse strain. Received: 12 January 1999 / Revised: 17 March 1999     

Assignment of the Ly-6--Ril-1--Sis--H-30--Pol-5/Xmmv-72--Ins-3--Krt-1--Int-1--Gdc-1 region to mouse chromosome 15

Previous work has demonstrated linkage between Ly-6, H-30, and a locus, Ril-1, that affects susceptibility to radiation-induced leukemia. Results of preliminary linkage analyses suggested further that the cluster might be linked to Ly-11 on the proximal portion of mouse chromosome 2. Using molecular probes to examine somatic cell lines and recombinant inbred and congenic strains of mice, we have re-evaluated these linkage relationships. A cloned genomic DNA fragment derived from a retroviral site has been used to define a novel locus, Pol-5, that is tightly linked to both H-30 and Ril-1 as shown by analysis of the B6.C-H-30 ^c congenic mouse strain. Following the segregation of the Pol-5 mouse-specific DNA fragment in a series of somatic cell hybrids carrying various combinations of mouse chromosomes on a rat or Chinese hamster background mapped Pol-5 to mouse chromosome 15. During the course of these studies, restriction fragment length polymorphisms were defined associated with several loci, including Pol-5, Ly-6, Sis, Ins-3, Krt-1, Int-1, and Gdc-1. Three of these loci, Sis, Int-1, and Gdc-1, have been previously mapped to chromosome 15 by others using somatic cell hybrids or isoenzyme analyses. Following the inheritance of these eight loci in recombinant inbred strains of mice allowed the definition of a linkage group on the chromosome with the order Ly-6-Ril-1--Sis--H-30--Pol-5--Ins-3--Krt-1--Int-1--Gdc-1. Analyses of alleles inherited as passengers in B6.C-H-30 ^c, C3H.B-Ly-6 ^b, and C57BL/6By-Eh/+ congenic mouse strains and in situ hybridization experiments support the above gene order and indicate further that the cluster is located on distal chromosome 15, with Ly-6 and Sis near Eh.Abbreviations A agouti - Abl cellular homolog of the Abelson leukemia virus oncogene - Ada adenosine deaminase - Ak-1 adenylate kinase-1 - AXB A/J × C57BL/6J recombinant inbred strain - B2m beta-2 microglobulin - BXA C57BL/6J × A/J recombinant inbred strain - BXD C57BL/6J × DBA/2J recombinant inbred strain - BXH C57BL/6J × C3H/HeJ recombinant inbred strain - CXB BALB/cBy × C57BL/6By recombinant inbred strain - DNA deoxyribonucleic acid - Eh hairy ears - Fpgs folypolyglutamyl synthetase - FXI fractionated x-irradiation - Gdc-1 glycerol phosphate dehydrogenase-1 - Il2r IL-2 receptor - Ins-3 a novel insulinlike gene - Int-1 mammary tumor integration site-1 - Itp inosine triphosphatase - Krt-1 the locus designated here includes a cluster of at least three keratin genes - LTR long terminal repeat - Ly lymphocyte - Lv-6 lymphocyte antigen-6 - Ly-11 lymphocyte antigen-11 - MIH minor histocompatibility - Myc cellular homolog of the Abelson leukemia virus oncogene; pa, pallid; - Pol-5 locus encoding retroviral polymerase-5 - RFLP restriction fragment length polymorphism - RI recombinant inbred mouse strains - Ril-1 radiation-induced leukemia susceptibility-1 locus - SDP strain distribution pattern - Sis cellular homolog of the simian sarcoma virus oncogene - SFFV spleen focus-forming virus - Tpi-1 triosephosphate isomerase-1 - Ve velvet     

Acid phosphatase-11, a tightly linked molecular marker for root-knot nematode resistance in tomato: from protein to gene,using PCR and degenerate primers containing deoxyinosine

With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus. The acid phosphatase-1 allozyme (APS-1¹), encoded by the Aps-1 ¹ allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots and suspension cells. Microsequencing of CNBr and tryptic peptides generated from APS-1¹ provided a partial amino acid sequence, which accounted for approximately 23% of the protein and revealed two stretches of homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acid residues. The partial amino acid sequence information enabled us to isolate a 2.4 kb genomic Aps-1 ¹ sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences. Synthesis of the 2.4 kb PCR product was specific for genomic templates carrying the L. peruvianum Aps-1 ¹ allele. Crucial to the priming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in which the number of different primer species was limited by incorporating deoxyinosine phosphate residues at three and four base ambiguities. In using cDNA as a template, a 490 bp sequence was obtained, indicating a high proportion of intron sequences in the 2.4 kb genomic Aps-1 ¹ sequence. The Aps-1 ¹ origin of the PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a small chromosomal region around the Aps-1/Mi loci.     

The Preparation of Congenic Strains of Tetrahymena1

SYNOPSIS. Congenic strains of syngen 1, Tetrahymena pyriformis, were produced by backcrossing the F¹ hybrid between inbred strains C2 and D to strain D in 12 consecutive backcrosses, with selection for certain C2 genes, and then using genomic exclusion to induce homozygosity. Six congenic strains of high breeding performance are available. Five differ from strain D in single genes at 5 different loci. The 6th strain differs at all 5 loci. Assuming the size of the Drosophila gene (4 × 10⁴ nucleotide pairs), we can calculate that strains differing from D by single genes have a heterozygous segment 3 genes long while The strain which differs from D by 5 genes has 5 heterozygous segments and 15 genes contributed from strain C2. A 40-fold increase in heterozygosity would be found with a gene size of 10³ nucleotide pairs. This means that in using these strains for biochemical work we must be aware that some genetic noise still remains.     

Production of congenic mouse strains carrying genomic intervals containing SLE-susceptibility genes derived from the SLE-prone NZM2410 strain

Systemic lupus erythematosus is inherited as a complex polygenic trait. Four genomic intervals containing major SLE-susceptibility loci were previously identified by interval mapping in the NZM2410 mouse model. In this paper, we utilized a marker-assisted selection protocol to produce four congenic mouse strains, each carrying an NZM2410-derived SLE-susceptibility interval on a C57BL/6-resistant background. Each strain carries only one susceptibility allele derived from this polygenic model and consequently can be used to characterize the specific component phenotypes contributed by individual SLE-susceptibility genes. We illustrate the efficacy of this approach with phenotypic data for one of our congenic strains, B6.NZMH2 ^z . Our results indicate that this single genomic interval from Chromosome (Chr) 17 of NZM2410 can mediate increased levels of IgG autoantibodies specific for chromatin and that, similar to results obtained in our original genetic cross, B6.NZMH2 ^z/b heterozygotes are more prone than B6.NZMH2 ^z homozygotes to the development of humoral autoimmunity to nuclear antigens. These results illustrate the feasibility of using congenic strains to dissect the complex pathogenic mechanisms that mediate polygenic SLE. These congenic strains will be valuable tools in the genetic analysis of SLE susceptibility. In future studies, these congenic strains will be interbred to produce bi- and tri-congenic strains in order to assess the role of genetic interactions in the expression of specific components of SLE pathogenesis. They will also be instrumental to the positional cloning and identification of the genes responsible for SLE susceptibility, via the production of congenic recombinants. Received: 1 September 1995 / Accepted: 20 December 1995     

Tracking genes of ecological relevance using a genome scan in two independent regional population samples of Arabis alpina

Understanding the genetic basis of adaptation in response to environmental variation is fundamental as adaptation plays a key role in the extension of ecological niches to marginal habitats and in ecological speciation. Based on the assumption that some genomic markers are correlated to environmental variables, we aimed to detect loci of ecological relevance in the alpine plant Arabis alpina L. sampled in two regions, the French (99 locations) and the Swiss (109 locations) Alps. We used an unusually large genome scan [825 amplified fragment length polymorphism loci (AFLPs)] and four environmental variables related to temperature, precipitation and topography. We detected linkage disequilibrium among only 3.5% of the considered AFLP loci. A population structure analysis identified no admixture in the study regions, and the French and Swiss Alps were differentiated and therefore could be considered as two independent regions. We applied generalized estimating equations (GEE) to detect ecologically relevant loci separately in the French and Swiss Alps. We identified 78 loci of ecological relevance (9%), which were mainly related to mean annual minimum temperature. Only four of these loci were common across the French and Swiss Alps. Finally, we discuss that the genomic characterization of these ecologically relevant loci, as identified in this study, opens up new perspectives for studying functional ecology in A. alpina, its relatives and other alpine plant species.     

Generation of CD4CreERT2 transgenic mice to study development of peripheral CD4‐T‐cells

After thymic emigration CD4‐T‐cells continue to differentiate into multiple effector and suppressor sublineages in peripheral lymphoid organs. In vivo analysis of peripheral CD4‐T‐cell differentiation has relied on animal models with targeted gene mutations. These are expressed either constitutively or conditionally after Cre mediated recombination. Available Cre transgenic strains to specifically target T‐cells act at stages of thymocyte development that precede thymic selection. Tracing gene functions in CD4‐T‐cell development after thymic exit becomes complicated when the targeted gene is essential during thymic development. Other approaches to conditionally modify gene functions in peripheral T‐cells involve infection of in vitro activated cells with Cre expressing lenti‐, retro‐, or adenoviruses, which precludes in vivo analyses. To study molecular mechanisms of peripheral CD4‐T‐cell differentiation in vivo and in vitro we generated transgenic mice expressing a tamoxifen inducible Cre recombinase (CreER^T2) under the control of the CD4 gene promoter. We show here that in CD4CreER^T2 mice Cre is inducibly and selectively activated in CD4‐T‐cells. Tamoxifen treatment both in vivo and in vitro results in efficient recombination of loci marked by LoxP sites. Moreover, this strain shows no abnormalities related to transgene insertion. Therefore it provides a valuable tool for studying gene function during differentiation of naïve peripheral CD4‐T‐cells into effector or suppressor sub‐lineages. genesis 50:908–913, 2012. © 2012 Wiley Periodicals, Inc.     

The Effect of Azidothymidine on Reactivation of DNA Synthesis in Macrophage Nuclei Contained in Heterokaryons

In heterokaryons, DNA synthesis is reactivated in macrophage nuclei only in the case of fusion with immortal cells. Assuming that telomerase is responsible for reactivation, the effect of its inhibitor azidothymidine (AZT) was studied in heterokaryons of mouse resident peritoneal macrophages and immortal 3T3 Swiss cells. AZT suppressed reactivation of DNA synthesis in macrophage nuclei and had no effect on DNA synthesis in 3T3 Swiss cell nuclei, suggesting that telomere structure is impaired in normal mouse macrophages.     

Development of gene-targeted SNP markers for genomic mapping in Pacific abalone Haliotis discus hannai Ino

Useful and novel DNA markers are needed for aquaculture genetics and breeding. In this study, we report the discovery and development of gene-targeted single nucleotide polymorphisms (SNPs) for genomic mapping in the Pacific abalone Haliotis discus hannai Ino. Single EST or EST-contigs from 66 genes that had positive BLASTx matches (E-value ≤ 1e-8) were used for polymerase chain reaction (PCR) amplification. PCR products from the two parents of one mapping family were directly sequenced, and 83 SNP loci were found from 17 genes. Allele-specific PCR (AS-PCR) was developed and optimized for genotyping of 11 SNP loci in 120 progeny of the mapping family. Nine of the loci conformed to the expected Mendelian ratio of 1:1 based on the χ² test (P > 0.05) and could potentially be used for linkage map construction. Our data also indicate that the sequencing of two parents may be a practical strategy for the discovery of informative SNPs for linkage mapping in a particular mapping population.     

Physical mapping of the MHC and grc by the pulse field electrophoresis

The development of the physical map of the major histocompatibility complex of the rat was undertaken using pulse field gel electrophoresis of fragments of genomic DNA from the BIL/2 (grc ⁺) and BIL/1 (grc ^–) strains obtained primarily from single and double digests with the enzymes Mlu I, Not I, and Sfi I and hybridized with a variety of mouse, rat, and human probes. Both strains are maintained by inbreeding the BIL heterozygote (forced heterozygosity; F31); hence, their differences lie almost entirely in the MHC-grc regions. The MHC-grc region was contained in five fragments of DNA comprising 3000–3200 kilobases (kb); thus, its size appears to be closer to that of the human MHC than to that of the mouse MHC. This didstance may be an underestimate of the size of the entire region, however, because the cluster of class I loci in the RT1.A region could not be defined in detail in this study. The most striking difference between the BIL/2 strain, which has normal growth and reproductive characteristics, and the BIL/1 strain, which has growth and reproductive defects and an enhanced susceptibility to chemical carcinogens, is a deletion of approximately 70 kb in the latter strain. The studies og grc ⁺ and grc ^– strain suggest that the phenotypic defects of the grc ^– stains may be due to the loss of genes that are normally present in this deleted region. Address correspondence and offprint request to: T. J. Gill III.     

QTL analyses of lineage-negative mouse bone marrow cells labeled with Sca-1 and c-Kit

Differences in the number of functionally and/or phenotypically defined bone marrow cells in inbred mouse strains have been exploited to map quantitative trait loci (QTL) that determine the variation in cell frequency. To extend this approach to the differences in the stem/progenitor cell compartment in CBA/H and C57BL/6 mice, we have exploited the resolution of flow cytometry and the power of QTL analyses in 124 F₂ mice to analyze lineage-negative (Lin⁻) bone marrow cells according to the intensity of labeling with Sca-1 and c-Kit. In the Lin⁻ Sca-1⁺ c-Kit⁺ enriched population, six QTL were identified: one significant and five suggestive. Whereas previous in vitro clonogenic, LTC-IC, day 35 CAFC, and flow cytometry each identified different QTL, our approach identified the same or very similar QTL at all three loci (chromosomes 1, 17, and 18) as well as QTL on chromosomes 6 and 10. In silico analyses implicate hematopoietic stem cell homing involving Cxcr4 and Cxcl12 as being the determining pathway. The mapping of the same or very similar QTL in independent studies using different assay(s) suggests a common genetic determinant, and thus reinforces the biological and genetic significance of the QTL. These data also suggest that mouse bone marrow cell subpopulations can be functionally, phenotypically, and genetically defined.     

Non Ionising Radiation as a Non Chemical Strategy in Regenerative Medicine: Ca2+-ICR “In Vitro” Effect on Neuronal Differentiation and Tumorigenicity Modulation in NT2 Cells

In regenerative medicine finding a new method for cell differentiation without pharmacological treatment or gene modification and minimal cell manipulation is a challenging goal. In this work we reported a neuronal induced differentiation and consequent reduction of tumorigenicity in NT2 human pluripotent embryonal carcinoma cells exposed to an extremely low frequency electromagnetic field (ELF-EMF), matching the cyclotron frequency corresponding to the charge/mass ratio of calcium ion (Ca²⁺-ICR). These cells, capable of differentiating into post-mitotic neurons following treatment with Retinoic Acid (RA), were placed in a solenoid and exposed for 5 weeks to Ca²⁺-ICR. The solenoid was installed in a μ-metal shielded room to avoid the effect of the geomagnetic field and obtained totally controlled and reproducible conditions. Contrast microscopy analysis reveled, in the NT2 exposed cells, an important change in shape and morphology with the outgrowth of neuritic-like structures together with a lower proliferation rate and metabolic activity alike those found in the RA treated cells. A significant up-regulation of early and late neuronal differentiation markers and a significant down-regulation of the transforming growth factor-α (TGF-α) and the fibroblast growth factor-4 (FGF-4) were also observed in the exposed cells. The decreased protein expression of the transforming gene Cripto-1 and the reduced capability of the exposed NT2 cells to form colonies in soft agar supported these last results. In conclusion, our findings demonstrate that the Ca²⁺-ICR frequency is able to induce differentiation and reduction of tumorigenicity in NT2 exposed cells suggesting a new potential therapeutic use in regenerative medicine.     

Identification of Neurofibromatosis 1 (NF1) Homologous Loci by Direct Sequencing, Fluorescence in Situ Hybridization, and PCR Amplification of Somatic Cell Hybrids

Using fluorescence in situ hybridization (FISH), we have identified seven NF1-related loci, two separate loci on chromosome 2, at bands 2q21 and 2q33-q34, and one locus each on five other chromosomes at bands 14q11.2, 15q11.2, 18p11.2, 21q11.2-q21, and 22q11.2. Application of PCR using NF1 primer pairs and genomic DNA from somatic cell hybrids confirmed the above loci, identified additional loci on chromosomes 12 and 15, and showed that the various loci do not share homology beyond NF1 exon 27b. Sequenced PCR products representing segments corresponding to NF1 exons from these loci demonstrated greater than 95% sequence identity with the NF1 locus. We used sequence differences between bona fide NF1 and NF1 -homologous loci to strategically design primer sets to specifically amplify 30 of 36 exons within the 5′ end of the NF1 gene. These developments have facilitated mutation analysis at the NF1 locus using genomic DNA as template.     

A Pdgf‐cCreERT2 knock‐in mouse model for tracing PDGF‐C cell lineages during development

PDGF‐C, a member of the platelet‐derived growth factor (PDGF) family, plays important roles in the development of craniofacial structures, the neural system, the vascular system, and tumors. PDGF‐C could also be required for the regulation of certain types of stem or progenitor cells as suggested by its expression in the regions where these cells are located. To further characterize the role of PDGF‐C in development, we generated a Pdgf‐c^CreERT2 mouse strain, in which a tamoxifen‐inducible Cre (CreERT2) cDNA was specifically targeted into the Pdgf‐c genomic locus and controlled by the endogenous Pdgf‐c regulatory elements. We also showed that Cre activity in this mouse strain could be specifically induced by tamoxifen, which allowed the fate of PDGF‐C‐expressing cells to be traced at various stages of development. Using this model system, we demonstrated for the first time that PDGF‐C‐expressing cells could be multipotent, generating multiple cell lineages required for the formation of the cerebellum. Therefore, the Pdgf‐c^CreERT2 mouse strain generated in this study will be a valuable transgenic tool for exploring the function of PDGF‐C in development and stem cell biology.     

Comparative analysis of the structure and chromosomal assignment ofCD1: an evidence for different evolutionary histories between classic CD1 and CD1D class genes

The non-MHC-encoded CD1 family has recently emerged as a novel antigen-presenting system that is distinct from MHC class I and class II molecules. In the present study, we determined the genomic structure of that rat CD1, and compared with those of other previously reported CD1 genes. Rat CD1 was extremely similar to mouse CD1 genes, especially to CD1D1. It is of interest that a tyrosine-based motif for endosomal localization, identified in the human CD1b cytoplasmic tail, was conserved in all CD1 molecules except for CD1a, that was encoded by a single short exon. Comparison of the overall exon-intron organization of CD1 genes revealed that the length of the introns was also characteristic to each of the two classes of CD1 genes; classic (CD1A, CD1B, CD1C and CD1E), and CD1D, which have been categorized by comparison of coding regions. These findings support a hypothesis that the two classes have different evolutionary histories. In contrast to the absence of the classic CD1 genes in rats and mice, the entire region of nonpolymorphic CD1D gene has been conserved through mammalian evolution. Furthermore, we determined chromosomal localization of rat CD1 gene using the fluorescence in situ hybridization method with several probes derived from genomic rat CD1 clones. Similar to human and mouse CD1, rat CD1 mapped outside the MHC loci despite the structural and functional resemblance to MHC. Conserved syntheny of chromosomal segments of RNO2 and MMU3 is implied.     

Genetic mapping of two DNA markers,D16Ros1 and D16Ros2, flanking the mutation site in the chakragati mouse,a transgenic insertional mutant

We present here the genetic mapping of two novel loci, D16Ros1 and D16Ros2, to mouse Chromosome (Chr) 16. The probes for these loci were genomic framents isolated from the chakragati mouse, a behavioural mutant resulting from insertional mutagenesis during the course of making transgenic mice. D16Ros1 and D16Ros2 were first mapped by recombinant inbred (RI) strain analysis and subsequently by the analysis of 145 progeny of two interspecific backcrosses between Mus domesticus and Mus spretus. These progeny had been typed for the centromere and this allowed mapping of D16Ros1 and D16Ros2 relative to the centromere. The other markers included in this study were Prm-1, Gap43 and Sod-1. The genetic map generated spanned 47.5 cM from the centromere to Sod-1, the most distal marker mapped here. The linkage data presented here should prove useful in mapping other loci relative to the centromere of Chr 16.     

Five genetic loci involved in the synthesis of acidic exopolysaccharides are closely linked in the genome of Rhizobium sp strain NGR234

Summary R-prime plasmids were constructed from a derivative of Rhizobium strain NGR234 (ANU280) and were shown to contain overlapping genomic DNA segments involved in biosynthesis of exopolysaccharides (EPS). The R-primes originally constructed carried the mutant allele from Tn5-induced EPS-deficient (Exo^–) mutant ANU2811. This plasmid-located mutant allele was dominant to the corresponding wild-type allele as merodiploid strains were Exo^–. Exo⁺ revertants occurred at a low rate (1×10^-7) and these were shown to result from double reciprocal recombination events, which led to the isolation of R-prime plasmids carrying functional wild-type exo alleles. R-prime plasmids that carry overlapping segments of DNA from parental strain ANU280 complemented 28 of the 30 group 2 Exo^– mutants of strain ANU280. Complementation of these Exo^– mutants also restored their symbiotic abilities of effective nodulation. Subsequent in vivo recombination between the wild-type alleles located on the R-prime and the corresponding mutated allele on the genome, was used to generate a new family of R-primes, which carried mutations in the exo genes. The 30 group 2 Exo^– mutants were classified into 7 distinct genetic groups based upon complementation and physical mapping data. Five of the seven exo loci were gentically linked and located on a 15-kb region of DNA. Mutations at two loci were dominant only when the mutations were R-prime plasmid-located while a mutation at a second locus was cis-dominant to two other exo loci. At least five genes involved in the synthesis of acidic exopolysaccharide synthesis have been identified.