A BAC-Based Integrated Linkage Map of the Silkworm \u3cem\u3eBombyx mori\u3c/em\u3e

Background: In 2004, draft sequences of the model lepidopteran Bombyx mori were reported using whole-genome shotgun sequencing. Because of relatively shallow genome coverage, the silkworm genome remains fragmented, hampering annotation and comparative genome studies. For a more complete genome analysis, we developed extended scaffolds combining physical maps with improved genetic maps. Results: We mapped 1,755 single nucleotide polymorphism (SNP) markers from bacterial artificial chromosome (BAC) end sequences onto 28 linkage groups using a recombining male backcross population, yielding an average inter-SNP distance of 0.81 cM (about 270 kilobases). We constructed 6,221 contigs by fingerprinting clones from three BAC libraries digested with different restriction enzymes, and assigned a total of 724 single copy genes to them by BLAST (basic local alignment search tool) search of the BAC end sequences and high-density BAC filter hybridization using expressed sequence tags as probes. We assigned 964 additional expressed sequence tags to linkage groups by restriction fragment length polymorphism analysis of a nonrecombining female backcross population. Altogether, 361.1 megabases of BAC contigs and singletons were integrated with a map containing 1,688 independent genes. A test of synteny using Oxford grid analysis with more than 500 silkworm genes revealed six versus 20 silkworm linkage groups containing eight or more orthologs of Apis versus Tribolium, respectively. Conclusion: The integrated map contains approximately 10% of predicted silkworm genes and has an estimated 76% genome coverage by BACs. This provides a new resource for improved assembly of whole-genome shotgun data, gene annotation and positional cloning, and will serve as a platform for comparative genomics and gene discovery in Lepidoptera and other insects

Introduction of GFP-Construct Into Medakafish Using The Particle Gun Method

Medakafish (Oryzias latipes) belongs to a group of small oviparous teleost fishes that inhabit fresh water in Asian countries including Japan. Experimental procedures to introduce foreign genes into medakafish have been reported using electroporation and/or microinjection. Microinjection is widely used in gene transfer experiments. However, it requires high technical sophistication. Electroporation can be easily applied to gene transfer experiments, but with a low efficiency. Therefore, we attempted to develop an experimental procedure to introduce a foreign gene without high technical sophistication but at reasonably high efficiency. A particle gun is an instrument used to introduce DNA that is coated on gold microcarrier particles directly into cells at high speed using high-pressure gas. Here we report a procedure to introduce a foreign gene into fertilized eggs of medakafish using the particle gun method. A plasmid construct with the green fluorescence protein (GFP) gene driven by the medakafish beta-actin gene promoter was successfully introduced into eggs, and the expression of GFP was observed in 20% of the primary transfectant fish. In addition, germ line transmission of GFP was observed in 13% of the GFP-positive primary transfectant.12th International Sympojium on Bioluminescence and Chemiluminescenc

Introduction of GFP-construct into medakafish using the particle gun method

Medakafish (Oryzias latipes) belongs to a group of small oviparous teleost fishes that inhabit fresh water in Asian countries including Japan. Experimental procedures to introduce foreign genes into medakafish have been reported using electroporation and/or microinjection. Microinjection is widely used in gene transfer experiments. However, it requires high technical sophistication. Electroporation can be easily applied to gene transfer experiments, but with a low efficiency. Therefore, we attempted to develop an experimental procedure to introduce a foreign gene without high technical sophistication but at reasonably high efficiency. A particle gun is an instrument used to introduce DNA that is coated on gold microcarrier particles directly into cells at high speed using high-pressure gas. Here we report a procedure to introduce a foreign gene into fertilized eggs of medakafish using the particle gun method. A plasmid construct with the green fluorescence protein (GFP) gene driven by the medakafish beta-actin gene promoter was successfully introduced into eggs, and the expression of GFP was observed in 20% of the primary transfectant fish. In addition, germ line transmission of GFP was observed in 13% of the GFP-positive primary transfectant.12th International Sympojium on Bioluminescence and Chemiluminescenc

Effect of SCID mutation on the occurrence of mouse Pc-1 (Ms6-hm) germline mutations.

Mouse Pc-1 (Ms6-hm) is a hypervariable minisatellite locus that is unstableduring intergenerational transmission. This hyper-instability of Pc-1 isuseful for detecting germline mutation using a small number of experimentalanimals, although its molecular mechanism has not yet been elucidated. Weexamined the effect of severe combined immune deficiency (SCID) mutation onthe spontaneous germline mutation at the Pc-1 locus using the CB17 mousestrain. Our results showed that the frequency of spontaneous germlinemutation at Pc-1 in the offspring of wild-type parents was 9.7%.In F1between SCID male and wild-type female, however, the frequency of germlinemutation was drastically increased to 42.3%. When SCID female mice were mated with wild-type male, the frequency of germline mutation in F1 was slightly increased to 13.6%. These results suggest that DNA protein kinase catalytic subunit (DNA-PKcs), deficiency of which causes SCID mutation, plays an important role in the stable transmission of a genome containing hypervariable tandem repeats to progeny in male germ cells

Introduction of a foreign gene into medakafish using the particle gun method

We developed a procedure to introduce a foreign gene into fertilized eggs ofmedakafish (Oryzias latipes) using the particle gun method, which is one ofthe easiest and most reliable techniques for gene transfer. A plasmidconstruct with the green fluorescence protein (GFP) gene driven by themedakafish beta-actin gene promoter was successfully introduced into eggs,and the expression of GFP was observed in 20% of the primary transfectant(chimera) fish. In addition, germ line transmission of GFP was observed in 13% of the GFP-positive primary transfectant fish. The new application described here should enable us to investigate gene expression using thefish model on a routine basis without high technical sophistication

A transgene and its expression profile are stably transmitted to offspring in transgenic medaka generated by the particle gun method

A particle gun is used in a potential method for introducing foreign genes into fish. In this paper, we report on the stable transmission of a transgene and its expression profile of the F4 generation in the transgenic medaka (Oryzias latipes). We established four transgenic strains, which contained a green fluorescent protein (GFP) gene controlled by a medaka P-actin promoter, using a particle gun. One more transgenic strain was also generated by microinjection for comparison. In all five strains, the founder was discovered to be mosaic for the transgene. However, from the F1 to F4 generations, transgenes and their expression profiles were stably inherited in the Mendelian manner. The expression profile was common among the five strains regardless of the method for gene transfer: GFP fluorescence became detectable at an early neurula stage. In this stage, the fluorescence was observed ubiquitously in most tissues. As somite developed, GFP fluorescence became intense only in the skeletal muscle and lens but it decreased in other tissues. In adult fish, an intense fluorescence was restricted in the skeletal muscle and lens, while a considerably weak fluorescence was observed in the brain, gill, heart, kidney, spleen, and ovary. From these results, it was concluded that the transgene and its expression profile were stably transmitted to offspring, and thus the particle gun is an effective method for transgenesis in spite of its easiness