Construction and characterization of a BAC library for functional genomics in Xenopus tropicalis.

Large insert genomic DNA libraries are useful resources for genomic studies. Although the genome of Xenopus tropicalis stands as the amphibian reference genome because it benefitted from large-scale sequencing studies, physical mapping resources such as BAC libraries are lagging behind. Here we present the construction and characterization of a BAC library that covers the whole X. tropicalis genome. We prepared this BAC library from the genomic DNA of X. tropicalis females of the Adiopodoume strain. We characterized BAC clones by screening for specific loci, by chromosomal localization using FISH and by systematic BAC end sequencing. The median insert size is about 110kbp and the library coverage is around six genome equivalents. We obtained a total of 163,787 BAC end sequences with mate pairs for 77,711 BAC clones. We mapped all BAC end sequences to the reference X. tropicalis genome assembly to enable the identification of BAC clones covering specific loci. Overall, this BAC library resource complements the knowledge of the X. tropicalis genome and should further promote its use as a reference genome for developmental biology studies and amphibian comparative genomics.

Localization of human coagulation factor VIII (hFVIII) in transgenic rabbit by FISH-TSA: identification of transgene copy number and transmission to the next generation.

For chromosomal localization of the hFVIII human transgene in F2 and F3 generation of transgenic rabbits, FISH-TSA was applied. A short cDNA probe (1250 bp) targeted chromosomes 3, 7, 8, 9 and 18 of an F2 male (animal 1-3-8). Two transgenic offspring (F3) revealed signal positions in chromosome 3 and chromosomes 3 and 7, respectively. Sequencing and structure analysis of the rabbit orthologous gene revealed high similarity to its human counterpart. Part of the sequenced cDNA (1310 bp) served as a probe for FISH-TSA analysis. The rabbit gene was localized in the q arm terminus of the X chromosome. This result is in agreement with reciprocal chromosome painting between the rabbit and the human. The presented FISH-TSA method provides strong signals without any interspecies reactivity.

Localization of the single copy gene Mdh2 on Xenopus tropicalis chromosomes by FISH-TSA.

A single copy gene, mitochondrial malate dehydrogenase 2 (Mdh2), was localized on Xenopus tropicalis chromosomes by fluorescence in situ hybridization coupled with tyramide signal amplification (FISH-TSA). The respective cDNA was cloned and sequenced. The labeled probe hybridized with a subcentromeric region of the long arms of homologous chromosomes 3. Results of comparison of the gene localization with previously mapped X. laevis paralogs strongly suggest a common evolutionary origin of chromosomes 3 and 8 in X. laevis and chromosome 3 in X. tropicalis. This is the first time that a single copy gene has been visualized on X. tropicalis chromosomes. The FISH-TSA method gives a strong signal with a 1-kb labeled probe.

Effect of protein supplement source on porcine oocyte maturation and subsequent embryonic development after parthenogenetic activation.

The aim of this study was to compare the effect of purified GPBoS and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after their parthenogenetic activation. COCs were obtained from dissected follicles and cultured for 18, 24, 30, 36, 42 and 48 h in M-199 medium either with GPBoS or FCS. After 24 h with GPBoS, 91% of oocytes reached MI stage while in the medium supplemented with FCS, only 29% of oocytes reached the same stage (P < 0.05). The majority of oocytes from the FCS group (61%) reached MI stage approximately 6 h later. In the time periods between 36 to 48 h both groups of oocytes reached the same stage of maturation. After 48 h of culture the oocytes with extruded polar bodies were activated by a single electric pulse and then cultured with 4 mM 6-DMAP. Activated oocytes were cultured in PZM-3 medium supplemented with 3 mg/ml of BSA. After 7 days, the development and the quality of embryos were evaluated. The results showed that the maturation of oocytes in the presence of GPBoS significantly increased their subsequent developmental ability when compared with FCS supplementation (27% vs. 19% of blastocysts, P < 0.05). However, differential staining revealed that once blastocysts were formed in either group, they had the same total cell number (40 vs. 41) and also the ICM/total cell ratio (0.27 vs. 0.29).

Xstir polymorphism and absence of sex linkage in Xenopus laevis ME2 gene.

A fragment of ME2 cDNA from exon 2 to exon 11 was sequenced and the sequence submitted to GenBank. Analysis of the intron, probably intron 13, revealed a polymorphism which is due to the presence of tandem repetitions of Xstir elements. Genetic analysis of the parents and the offspring showed a standard distribution of intron variants. This distribution was not dependent on sex. We conclude, contrary to previous reports, that the ME2 gene is not linked to sex. Consequently, the Xstir polymorphism can be used as a tool for genetic analysis.

The c-SRC1 gene visualized by in situ hybridization on Xenopus laevis chromosomes.

Fluorescent in situ hybridization followed by tyramide signal amplification was used to map the site of the c-SRC1 gene on XENOPUS LAEVIS chromosomes. Positive results were obtained with a cDNA probe of about 1 kb. The c-SRC1 gene is located in the subcentromeric region in the long arm of one of the acrocentric chromosomes of the G category (classified according to Graf and Kobel, 1991). The c-SRC1 gene and the XENOPUS major histocompatibility complex (MHC) 1b locus, which consists of 20 tandemly arranged gene copies, are situated on different chromosomes of the G category.