FLPe functions in zebrafish embryos

To assay the efficiency of the FLP/FRT site-specific recombination system in Danio rerio, a construct consisting of a muscle-specific promoter driving EGFP flanked by FRT sites was developed. FLPe capped RNA was microinjected into transgenic single cell stage zebrafish embryos obtained by crossing hemizygous transgenic males with wild-type females. By 48 h post fertilization (hpf), the proportion of embryos displaying green fluorescence following FLPe RNA microinjection was significantly lower (7.7%; P < 0.001) than would be expected from a cross in the absence of the recombinase (50%). Embryos that retained fluorescence displayed marked mosaicism. Inheritance of the excised transgene in non-fluorescent, transgenic embryos was verified by PCR analysis and FLPe-mediated recombination was confirmed by DNA sequencing. Sperm derived from confirmed transgenic males in these experiments was used to fertilize wild-type eggs to determine whether germline excision of the transgene had occurred. Clutches sired by FLPe-microinjected males contained 0–4% fluorescent embryos. Transgenic males that were phenotypically wild-type produced no fluorescent progeny, demonstrating complete excision of the transgene from their germline. FLPe microinjected males that retained some fluorescent muscle expression produced a small proportion of fluorescent offspring, suggesting that in mosaic males not all germline cells had undergone FLPe-mediated transgene excision. Our results show that FLPe, which is derived from Saccharomyces cerevisiae, is an efficient recombinase in zebrafish maintained at 28.5°C

Phenotypic and genotypic diversity of wine yeasts used for acidic musts

The aim of this study was to examine the physiological and genetic stability of the industrial wine yeasts Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum under acidic stress during fermentation. The yeasts were sub-cultured in aerobic or fermentative conditions in media with or without l-malic acid. Changes in the biochemical profiles, karyotypes, and mitochondrial DNA profiles were assessed after minimum 50 generations. All yeast segregates showed a tendency to increase the range of compounds used as sole carbon sources. The wild strains and their segregates were aneuploidal or diploidal. One of the four strains of S. cerevisiae did not reveal any changes in the electrophoretic profiles of chromosomal and mitochondrial DNA, irrespective of culture conditions. The extent of genomic changes in the other yeasts was strain-dependent. In the karyotypes of the segregates, the loss of up to 2 and the appearance up to 3 bands was noted. The changes in their mtDNA patterns were much broader, reaching 5 missing and 10 additional bands. The only exception was S. bayanus var. uvarum Y.00779, characterized by significantly greater genome plasticity only under fermentative stress. Changes in karyotypes and mtDNA profiles prove that fermentative stress is the main driving force of the adaptive evolution of the yeasts. l-malic acid does not influence the extent of genomic changes and the resistance of wine yeasts exhibiting increased demalication activity to acidic stress is rather related to their ability to decompose this acid. The phenotypic changes in segregates, which were found even in yeasts that did not reveal deviations in their DNA profiles, show that phenotypic characterization may be misleading in wine yeast identification. Because of yeast gross genomic diversity, karyotyping even though it does not seem to be a good discriminative tool, can be useful in determining the stability of wine yeasts. Restriction analysis of mitochondrial DNA appears to be a more sensitive method allowing for an early detection of genotypic changes in yeasts. Thus, if both of these methods are applied, it is possible to conduct the quick routine assessment of wine yeast stability in pure culture collections depositing industrial strains

FLP Recombinase-Mediated Site-Specific Recombination in Silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species

Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review

The advantages of phage Mu transposition-based systems for the chromosomal editing of plasmid-less strains are reviewed. The cis and trans requirements for Mu phage-mediated transposition, which include the L/R ends of the Mu DNA, the transposition factors MuA and MuB, and the cis/trans functioning of the E element as an enhancer, are presented. Mini-Mu(LR)/(LER) units are Mu derivatives that lack most of the Mu genes but contain the L/R ends or a properly arranged E element in cis to the L/R ends. The dual-component system, which consists of an integrative plasmid with a mini-Mu and an easily eliminated helper plasmid encoding inducible transposition factors, is described in detail as a tool for the integration/amplification of recombinant DNAs. This chromosomal editing method is based on replicative transposition through the formation of a cointegrate that can be resolved in a recombination-dependent manner. (E-plus)- or (E-minus)-helpers that differ in the presence of the trans-acting E element are used to achieve the proper mini-Mu transposition intensity. The systems that have been developed for the construction of stably maintained mini-Mu multi-integrant strains of Escherichia coli and Methylophilus methylotrophus are described. A novel integration/amplification/fixation strategy is proposed for consecutive independent replicative transpositions of different mini-Mu(LER) units with “excisable” E elements in methylotrophic cells

Transposition-based method for the rapid generation of gene-targeting vectors to produce Cre/Flp-modifiable conditional knock-out mice

Peer reviewe

Impact of selenium enrichment on seed potato tubers

The aim of this study was to investigate the effect of Se enrichment on the growth of sprouts and growth vigour of seed potatoes (Solanum tuberosum L.) stored for 2 to 8 months. Our results showed that Se did not affect the duration of dormancy. At the high addition levels (0.075 and 0.9 mg kg-1 quartz sand), Se had some positive effects on the growth of sprouts. The peak sprouting capacity was reached after 8 months of storage. The highest Se enrichment of tubers had some positive effect on the free putrescine content in sprouts. However, the better growth of sprouts was not consistent with the growth vigour of the seed tubers and yield produced. Selenium had no significant effect on the malondialdehyde (MDA) or on the concentration of soluble sugars and starch. No significant effect of added Se on the early growth, stem and tuber numbers and yield parameters was observed. Irrespective of the level of Se added, the highest yield was harvested from plants produced with seed tubers stored for 6 months. Our results indicate that Se had some positive effects on the growth of sprouts, but it had no consistent effect on the growth vigour of seed tubers.;Suomessa perunaa joudutaan varastoimaan pitkaan. Varastoinninaikana siemenperunassa tapahtuu fysiologisia ja biokemiallisia muutoksia, jotka johtavat hiljalleen fysiologiseen vanhenemiseen ja sita kautta mukulan elinvoiman ja sadonmuodostuskyvyn alenemiseen. Seleeni (Se) on valttamaton alkuaine ihmisten ja elainten terveydelle. Viljelykasvien ei ole katsottu valttamatta tarvitsevan seleenia. Kuitenkin pienen seleenilisayksen on havaittu lisaavan kasvien antioksidatiivista kapasiteettia, parantavan kasvien kasvua, satoa ja laatua seka hidastavan kasvien vanhenemista. Helsingin yliopistossa Soveltavan biologian laitoksella tutkittiin voiko seleeni edistaa siemenperunan elinvoiman sailymista seka hidastaa vanhenemista varastoinnin aikana. Seleenirikastetut siemenperunat tuotettiin edellisena kasvukautena kasvihuoneessa kasveissa, jotka kasvatettiin nousevilla seleenimaarilla (0, 0.0035, 0.01, 0.075 ja 0.9 mg Se kg-1) lannoitetussa kvartsihiekassa, joilla siemenperunoiden seleenipitoisuudet olivat keskimaarin 0.01, 0.09, 0.20, 1.33 and 16 Êg Se g-1 kuivapainoa, tassa jarjestyksessa. Seleenin vaikutusta siemenperunan itujen kasvuun tutkittiin maarittamalla itujen lukumaara ja itamiskapasiteetti. Iduista maaritettiin vapaiden polyamiinien pitoisuus. Malondialdehydin (MDA) ja liukoisten sokereiden kertyminen sekä tärkkelyksen hajoaminen määritettiin siemenperunoista varastoinnin aikana. Siemenperunoiden elinvoima tutkittiin kasvihuonekokeissa määrittämällä taimettumisaika, kukinnan alkamisaika, pääversojen lukumäärä sekä kasvikohtaisesti sato, mukulalukumäärä ja mukulapaino. Tutkimuksen tulokset osoittivat, että korkeimmat seleenilisäykset (0.075 ja 0.9 mg kg-1) paransivat itämiskapasiteettia eli suurempi osa olemassa olevista iduista oli lähtenyt kasvuun. Korkeimmalla seleenirikastuksella oli positiivinen vaikutus itujen vapaan putreskiinin pitoisuuteen. Lisääntynyt itujen kasvu ei ollut yhteydessä siemenperunoiden elinvoimaan eli tuotetun sadon määrään. Seleenirikastuksella ei ollut vaikutusta perunoiden MDA:n eikä liukoisten sokereiden kertymiseen eikä tärkkelyksen hajoamiseen. Korkein sato tuotettiin kuusi kuukautta varastoidulla siemenperunalla, riippumatta seleenilisäyksestä. Kahdeksan kuukautta varastoidut siemenperunat sen sijaan tuottivat alhaisemman sadon, mikä viittaa siihen, että siemenperunan optimaalinen ikä oli jo ohitettu. Tulokset osoittavat, että seleenirikastuksella ei ollut selvää siemenperunoiden vanhenemista hidastavaa eikä niiden elinvoimaa edistävää vaikutusta