We have recently Alpelisib isolated antimicrobial compound-producing strains from oyster haemolymph, suggesting that microbiota may confer a health benefit on the host (Defer et al., 2013). In this study, we have explored the cultivable haemolymph-associated bacteria in four bivalves (oyster, clam, mussel and scallop) for their antimicrobial activity. The most potent ones were also investigated for hemocyte cytotoxicity. Results are clearly in

line with the hologenome concept. Moreover, they suggest that haemolymph-associated bacteria are a potential source of aquaculture probiotics. To limit the impact of anthropic pressure, bivalve specimens were collected by deep-sea diving in the Glenan Archipelago (47°43′N, 4°01′W, WGS84 system), a Natura 2000 TSA HDAC research buy area (FR5300023), during winter

2009 and spring 2010. Selected species were the oyster (Crassostrea gigas), the blue mussel (Mytilus edulis), the scallop (Pecten maximus), and the pink clam (Tapes rhomboides). Haemolymph of each individual was collected aseptically by inserting a 25-gauge needle attached to a 1-mL syringe directly into the adductor muscle. For C. gigas, haemolymph was collected from the pericardium. A volume ranging from 0.5 to 1 mL of haemolymph was drawn from each mollusc and placed in ice to prevent the hemocyte aggregation. Each sample was microscopically examined to check the presence of healthy hemocytes. Checked haemolymph (50 μL) was spread onto Marine agar Petri dishes using a Wasp® automated spiral plater (AES Lab). Plates were further incubated for 72 h at 18 °C. To isolate as many different bacteria as possible, 1–10 macroscopically distinguishable colonies were picked and subcultured in Marine Broth for 48 h at 18 °C with shaking (100 r.p.m.). Bacterial purity was assessed Ketotifen by streaking on Marine Agar. For long-term storage, sterile glycerol was added to 1 mL bacterial culture (25% v/v) in cryogenic vials that were stored at −80 °C. Cell-free supernatants coming from culturable haemolymph-associated bacteria were assayed for antibacterial activity against a panel of 12 aquaculture pathogens (Table 1).

After growth (72 h, 18 °C, 100 r.p.m.), the culture supernatant (1 mL) was collected by centrifugation (6000 g for 10 min at 4 °C) and filtration (0.22 μm, SFCA serum Filter Unit, Nalgene). To detect antibacterial activity, the well-diffusion method was used (Wiegand et al., 2008; Defer et al., 2013). Specific agar medium according to bacterial target was inoculated with an 8-h-old culture broth of the indicator strain to a bacterial concentration of 1.106 CFU mL−1. Wells (diameter 4 mm) were punched into the agar medium and cell-free supernatants (20 μL) or controls (Marine Broth for negative control and polymyxin B sulphate and Nisaplin® at 1 mg mL−1 as positive control against respectively Gram-negative and Gram-positive target bacteria) were created.

Previously, we classified three factors (OmpR, RstA and IHF) as activators and two factors (CpxR and H-NS) as repressors, and found novel modes of their interplay. Here we describe an as yet uncharacterized regulator, MlrA, that has been suggested to participate in control of curli formation. Based on both in vivo and in vitro analyses, we identified MlrA as a positive factor of the csgD promoter by directly binding to its upstream region (−113 to −146) with a palindromic sequence

of AAAATTGTACA(12N)TGTACAATTTT between the binding sites of two activators, IHF and OmpR. The possible interplay between three activators was analysed in detail. Under stressful conditions in nature, planktonic single-cell Escherichia coli transforms into multicellular biofilm through adhesion to solid surfaces and cell–cell interactions using extracellular matrix compounds Selleck Afatinib such as cellulose and curli fimbriae (Prigent-Combaret Dabrafenib in vitro et al., 2000; Chapman et al., 2002; Beloin et al., 2008; Gualdi et al., 2008; Wood, 2009). The synthesis of csgBA-encoded curli is induced at low temperatures and

under low osmolarity during stationary phase growth (Barnhart & Chapman, 2006). Expression of csgBA is under the control of a positive regulator, CsgD, which is also involved in the regulation of cellulose production and peptidase synthesis (Prigent-Combaret et al., 2001; Brombacher et al., 2003, 2006; Chirwa & Herrington, 2003; Gerstel & Romling,

2003). In concert with the regulatory role of CsgD as the master regulator of biofilm formation under stressful conditions, the major sigma RpoD and stationary phase-specific RpoS participate in transcription initiation from two promoters of the csgD operon (Robbe-Saule et al., 2006; Gualdi et al., 2007; Ogasawara et al., 2007a, 2010). Furthermore, a number of transcription factors are involved in the regulation of the csgD promoter, including CpxR (Jubelin et al., 2005), Crl (Bougdour et al., 2004), H-NS (Gerstel et al., 2003), IHF (Gerstel et al., 2003, 2006), OmpR (Vidal et al., 1998; Gerstel et al., 2003, 2006), RstA (Ogasawara et al., 2007a), MlrA (Brown et al., 2001), RcsB (Ferrieres & Clarke, 2003; Vianney et al., Lonafarnib mouse 2005) and CRP (Zheng et al., 2004). On the basis of these observations, the csgD promoter is now recognized as one of the most complex promoters in E. coli (Ishihama, 2010). As an initial step toward understanding the regulatory mechanisms of the csgD promoter by a number of transcription factors, we have classified some of these transcription factors into positive and negative regulators (Ogasawara et al., 2010). In addition, we and others have analysed the pair-wise interplay between RpoS and Crl (Robbe-Saule et al., 2006), IHF and H-NS (Gerstel et al., 2003; Ogasawara et al., 2010), OmpR and IHF (Gerstel et al.

These four sequence blocks were separated selleck chemicals by a variable to a certain degree among the plasmids 10-mer sequence that was identical for each plasmid. Of note, the same 10-mer sequence could also be found preceding the first 12-mer block. DNA folding simulations for pREN

ori revealed a putative hairpin in the variable region and two identical stem loops in the iteron region (Fig. 3b). Similar secondary structure organizations could also be detected in the oris of all other plasmids (data not shown). While the significance of these structures remains to be investigated, it is important to state that the similarity in secondary structures among the plasmids is clearly driven by sequence conservation (Fig. 3a). The overall architecture of pREN was assessed in comparison with that of other members of the pUCL287 family of plasmids. Interestingly, while the replication backbone of pREN (ori and repA) was highly conserved (data not shown), blastn queries returned only two hits showing identity over the entire plasmid sequence, i.e. pLB925A03 and Nutlin-3a supplier pLJ42. pLB925A03 carries seven orfs on its 8881 bp sequence, consisting of two genes (repA and repB) involved in the replication process, three genes for mobilization and two

unknown genes. pLJ42 (5529 bp in length) encodes a replication (RepA) and three mobilization (MobA, MobB and MobC) proteins. We synchronized all three plasmid annotations so as to start from the first nucleotide of the repA gene in order to perform full-length Bacterial neuraminidase plasmid sequence alignments (Fig. 4). This comparative mapping of plasmids demonstrated that they share a common organization not only in their replication backbone (repA-orf2 operon and the ori regions) but also in the mobilization backbone. The three consecutive mob genes showed a high degree of identity among the plasmids, with the exception of pREN, which, due to the frameshift mutation mentioned earlier, had its mobA gene disrupted in two truncated pseudogenes. This organization of the replication and mobilization elements seems to be unique

within the pUCL287 family. According to our analysis, only pREN and pLJ42 possess the basal backbone for this type of plasmids, because an insertion of approximately 4500 bp was evident downstream of the mob genes for plasmid pLB925A03. Furthermore, the phylogeny of RepA, MobC and MobA was surveyed. MobB was excluded from this analysis because it could be detected in only five other bacteria, as mentioned earlier. In the case of MobA, the two truncated proteins of pREN were also omitted from the phylogenetic trees and therefore all conclusions presented below were based on the MobA sequence of plasmid pLB925A03. RepA of pREN clustered with the respective proteins of other Lactobacillus plasmids (Fig. 5a) and a clear relation of this cluster with several enterococci replication initiation proteins was observed.

These four sequence blocks were separated Venetoclax in vitro by a variable to a certain degree among the plasmids 10-mer sequence that was identical for each plasmid. Of note, the same 10-mer sequence could also be found preceding the first 12-mer block. DNA folding simulations for pREN

ori revealed a putative hairpin in the variable region and two identical stem loops in the iteron region (Fig. 3b). Similar secondary structure organizations could also be detected in the oris of all other plasmids (data not shown). While the significance of these structures remains to be investigated, it is important to state that the similarity in secondary structures among the plasmids is clearly driven by sequence conservation (Fig. 3a). The overall architecture of pREN was assessed in comparison with that of other members of the pUCL287 family of plasmids. Interestingly, while the replication backbone of pREN (ori and repA) was highly conserved (data not shown), blastn queries returned only two hits showing identity over the entire plasmid sequence, i.e. pLB925A03 and SCH727965 ic50 pLJ42. pLB925A03 carries seven orfs on its 8881 bp sequence, consisting of two genes (repA and repB) involved in the replication process, three genes for mobilization and two

unknown genes. pLJ42 (5529 bp in length) encodes a replication (RepA) and three mobilization (MobA, MobB and MobC) proteins. We synchronized all three plasmid annotations so as to start from the first nucleotide of the repA gene in order to perform full-length Thiamet G plasmid sequence alignments (Fig. 4). This comparative mapping of plasmids demonstrated that they share a common organization not only in their replication backbone (repA-orf2 operon and the ori regions) but also in the mobilization backbone. The three consecutive mob genes showed a high degree of identity among the plasmids, with the exception of pREN, which, due to the frameshift mutation mentioned earlier, had its mobA gene disrupted in two truncated pseudogenes. This organization of the replication and mobilization elements seems to be unique

within the pUCL287 family. According to our analysis, only pREN and pLJ42 possess the basal backbone for this type of plasmids, because an insertion of approximately 4500 bp was evident downstream of the mob genes for plasmid pLB925A03. Furthermore, the phylogeny of RepA, MobC and MobA was surveyed. MobB was excluded from this analysis because it could be detected in only five other bacteria, as mentioned earlier. In the case of MobA, the two truncated proteins of pREN were also omitted from the phylogenetic trees and therefore all conclusions presented below were based on the MobA sequence of plasmid pLB925A03. RepA of pREN clustered with the respective proteins of other Lactobacillus plasmids (Fig. 5a) and a clear relation of this cluster with several enterococci replication initiation proteins was observed.

However, media composition and incubation temperature can affect dye affinity and impose limitations on Selleck Osimertinib red phenotype detection by this method. In this study, we compared different Shiga toxin-producing E. coli for CR affinity and biofilm formation under different media/temperature conditions. We found strain and serotype differences in CR affinities and biofilm formation, as well as temperature and

media requirements for maximum CR binding. We also constructed strains with deletions of curli and/or cellulose genes to determine their contributions to the phenotypes and identified two O45 strains with a medium-dependent induction of cellulose. “
“The oxalate–carbonate pathway (OCP) leads to a potential carbon sink in terrestrial environments. This process is linked to the activity of oxalotrophic bacteria. Although isolation

and molecular characterizations are used to study oxalotrophic bacteria, these approaches do not give information on the active oxalotrophs present in soil undergoing the OCP. The aim of this study was to assess the diversity of active oxalotrophic bacteria in soil microcosms using the Bromodeoxyuridine (BrdU) DNA labeling technique. Soil was collected near GSK-3 beta pathway an oxalogenic tree (Milicia excelsa). Different concentrations of calcium oxalate (0.5%, 1%, and 4% w/w) were added to the soil microcosms and compared with an untreated control. After 12 days of incubation, a maximal pH of 7.7 was measured for microcosms with oxalate (initial pH 6.4). At this time point, a DGGE profile of the frc gene was performed Methocarbamol from BrdU-labeled soil DNA and unlabeled soil DNA. Actinobacteria (Streptomyces- and Kribbella-like sequences),

Gammaproteobacteria and Betaproteobacteria were found as the main active oxalotrophic bacterial groups. This study highlights the relevance of Actinobacteria as members of the active bacterial community and the identification of novel uncultured oxalotrophic groups (i.e. Kribbella) active in soils. “
“Natural resistance of wheat plants to wheat sharp eyespot is inadequate, and new strategies for controlling the disease are required. Biological control is an alternative and attractive way of reducing the use of chemicals in agriculture. In this study, we investigated the biocontrol properties of endophytic bacterium Bacillus cereus strain 0–9, which was isolated from the root systems of healthy wheat varieties. The phosphotransferase system is a major regulator of carbohydrate metabolism in bacteria. Enzyme I is one of the protein components of this system. Specific disruption and complementation of the enzyme I-coding gene ptsI from B. cereus was achieved through homologous recombination. Disruption of ptsI in B. cereus caused a 70% reduction in biofilm formation, a 30.4% decrease in biocontrol efficacy, and a 1000-fold reduction in colonization. The growth of ΔptsI mutant strain on G-tris synthetic medium containing glucose as the exclusive carbon source was also reduced.

However, media composition and incubation temperature can affect dye affinity and impose limitations on Angiogenesis inhibitor red phenotype detection by this method. In this study, we compared different Shiga toxin-producing E. coli for CR affinity and biofilm formation under different media/temperature conditions. We found strain and serotype differences in CR affinities and biofilm formation, as well as temperature and

media requirements for maximum CR binding. We also constructed strains with deletions of curli and/or cellulose genes to determine their contributions to the phenotypes and identified two O45 strains with a medium-dependent induction of cellulose. “
“The oxalate–carbonate pathway (OCP) leads to a potential carbon sink in terrestrial environments. This process is linked to the activity of oxalotrophic bacteria. Although isolation

and molecular characterizations are used to study oxalotrophic bacteria, these approaches do not give information on the active oxalotrophs present in soil undergoing the OCP. The aim of this study was to assess the diversity of active oxalotrophic bacteria in soil microcosms using the Bromodeoxyuridine (BrdU) DNA labeling technique. Soil was collected near E7080 manufacturer an oxalogenic tree (Milicia excelsa). Different concentrations of calcium oxalate (0.5%, 1%, and 4% w/w) were added to the soil microcosms and compared with an untreated control. After 12 days of incubation, a maximal pH of 7.7 was measured for microcosms with oxalate (initial pH 6.4). At this time point, a DGGE profile of the frc gene was performed Sitaxentan from BrdU-labeled soil DNA and unlabeled soil DNA. Actinobacteria (Streptomyces- and Kribbella-like sequences),

Gammaproteobacteria and Betaproteobacteria were found as the main active oxalotrophic bacterial groups. This study highlights the relevance of Actinobacteria as members of the active bacterial community and the identification of novel uncultured oxalotrophic groups (i.e. Kribbella) active in soils. “
“Natural resistance of wheat plants to wheat sharp eyespot is inadequate, and new strategies for controlling the disease are required. Biological control is an alternative and attractive way of reducing the use of chemicals in agriculture. In this study, we investigated the biocontrol properties of endophytic bacterium Bacillus cereus strain 0–9, which was isolated from the root systems of healthy wheat varieties. The phosphotransferase system is a major regulator of carbohydrate metabolism in bacteria. Enzyme I is one of the protein components of this system. Specific disruption and complementation of the enzyme I-coding gene ptsI from B. cereus was achieved through homologous recombination. Disruption of ptsI in B. cereus caused a 70% reduction in biofilm formation, a 30.4% decrease in biocontrol efficacy, and a 1000-fold reduction in colonization. The growth of ΔptsI mutant strain on G-tris synthetic medium containing glucose as the exclusive carbon source was also reduced.

Relevant characteristics of the bacterial strains, bacteriophage and plasmids used in this study are described in Table 1. Routine cell growth was carried out at 37 °C in Luria–Bertani (LB) medium supplemented with antibiotics as appropriate. Luria–Bertani medium and LB agar (1.8% agar) were prepared according to Miller (1972). Antibiotics were added as required: ampicillin (100 μg mL−1), kanamycin (40 μg mL−1) and chloramphenicol (20 μg mL−1). The enzymes for cloning were supplied by Fermentas. Hybrid plasmids and vectors Doramapimod nmr were isolated using a kit from Qiagen. Chromosomal DNA was isolated from the cells at late exponential phase of growth; the cells were lysed with lysozym and sodium dodecyl sulphate

and the lysate was then treated with phenol with subsequent DNA sedimentation in ethanol. Restriction, ligation of DNA fragments, electrophoresis in agarose gel, isolation of DNA fragments from the gel by electroelusion and transformation of calcium cells were performed in E. coli as described (Sambrook et al., 1989). The plasmid pTLΔHindIII was obtained by treatment of pKLH53.1 with HindIII and subsequent ligation. The HindIII fragment of 2.5 kbp

and HindIII-ClaI fragment from the mer operon of Tn5053 were cloned in pUC19 under the lac promoter: pTL2.5 (2.5-kbp HindIII fragment) and pTLHindIII-ClaI (HindIII-ClaI fragment). The fragment tniA,B,Q Tn5053 (2.3 kbp) was cloned in pUC19 under the lac promoter (pTLORF-5). Hybrid plasmid pSMΔORF-5 was learn more obtained by eliminating the DNA between the Eco47III sites within the orf-5 much gene in pTLORF-5 (see Fig. 2). In pORF-5, a 483-bp fragment from the tniA gene was cloned in pUC19 under the lac promoter (see Fig. 2). The DNA fragment containing the gene orf-5 was amplified by PCR using the following primers: Tn5053dir, 5′-GCAGAGGGTGACGGCCGGATGG-3′; Tn5053rev, 5′-CACGGCGATGCAGATGATCCACG-3′ and plasmid pKLH53.1 DNA as a template.

Amplification was carried out at the conditions recommended by the manufacturer. The amplification product was purified by electrophoresis and cloned in T-vector pTZ57R. A 483-bp fragment was then recloned into pUC19 at XbaI and BamHI restriction sites to construct pORF-5. For the other plasmid constructs of the pKLH series see Kholodii et al. (1995). The antirestriction activity of plasmid was defined as the efficiency of plating (EOP) of unmodified phage λ.0 on the experimental (plasmid-bearing) strain divided by the EOP on the plasmidless restricting strain (Delver et al., 1991). The EOP (in Table 2 designated К) was calculated as: phage titre on the restricting strain (NK114)/phage titre on a nonrestricting strain (TG-1). Unmodified phages, denoted by λ.0, were grown on E. coli TG-1 r−m−, which lost restriction and modification functions. All assays were performed in triplicate and at least 50 phage plaques per plate per experiment were counted.

Side effects were recorded individually and then categorised as being ‘significant’ or ‘minor’. A significant side effect was defined as a potentially life-threatening adverse reaction. Examples were mortality, inability to maintain an airway

or desaturations not corrected by head movements. Minor side effects were defined as any reported adverse events that were non-life-threatening. Examples of minor side effects were more difficult to subcategorise, principally due to an inconsistent use of terminology in studies. All have been reported. Data related to the effectiveness of the sedative were not collected. 4. Types of study: Allocation concealment, patient, operator or assessor blinding were not used as entry criteria for this review. Evidence was ranked according to its quality, and the ranking was as follows (highest first): Randomised controlled clinical trials of effectiveness Z-VAD-FMK datasheet and randomised controlled clinical trials looking at adverse outcomes Non-randomised studies. Prospective or retrospective observational studies (including case reports) Reference books and databases describing

adverse effects as listed in Chapter 14 of the Cochrane Review Handbook[6]. The search for RCTs was modelled on that used by Matharu and Ashley[7] in their effectiveness review in 2005. This version was used as the updated review Pembrolizumab excludes crossover trials. The search for any other non-randomised studies used a combination of controlled vocabulary and free text terms based on the search strategy as described in Chapter 14 of the Cochrane Handbook[6]. See Fig. 1 for Medline search, Fig. 2 for Embase search [MEDLINE (OVID), 1950 to November 2011 week 1; EMBASE (OVID) 1947–2011 November 8]. This was then supplemented by a further free text search as recommended in Chapter 14 of the Cochrane Handbook[6]. In addition, reference books and regulatory authorities were also searched for reports on oral midazolam using the website search engine and the free text term ‘midazolam’ (full list in Fig. 3)[8-11]. Specialist drug information databases were not searched due to subscription costs and as their usefulness

or additional yield have yet to be formally evaluated in the systematic review setting. The following journals were identified Janus kinase (JAK) as being important to be hand searched for this review: International Journal of Paediatric Dentistry, Pediatric Dentistry, Journal of American Dental Association, Anesthesia Progress. The journals were hand searched by the review authors for the period January 2000 to November 2011. The reference lists of all eligible trials were checked for additional studies. The search attempted to identify all relevant studies irrespective of language. Non-English papers were translated where possible. Results from these searches were combined together using Reference Manager (Thomson Corp, Carlsbad, CA, USA). The recommended adverse effects search terms as described by Loke et al.

An EGFP-positive Purkinje cell whose soma was located at a distance more than one soma away from the Purkinje cell layer, defined by the rest of the EGFP-negative Purkinje cells, was counted as ‘mislocalized’. Statistical significance was defined by the χ2 test. For the statistical analysis of electrophysiological results, the Mann–Whitney U-test www.selleckchem.com/products/ABT-263.html was applied. Previous studies demonstrated that mouse Purkinje cells arise from the ventricular zone facing the fourth ventricle around E10–E13 (Miale & Sidman, 1961; Wang & Zoghbi, 2001; Hashimoto & Mikoshiba, 2003). Thus, to develop an IUE method for Purkinje cells, a plasmid

encoding EGFP under the control of the CAG promoter (CAG-EGFP) was injected http://www.selleckchem.com/products/Bortezomib.html into the fourth ventricle of E10.5, E11.5 or E12.5 mice. To transfect Purkinje cell precursors, the electrodes were placed diagonally across the fourth ventricle with the anode above the cerebellar primordium at an angle of 90° or more to the targeted side of the upper rhombic lip (Fig. 1A

and Supporting Information, Fig. S1), and 33-V electrical pulses were applied five times (Fig. 1A). We observed bright EGFP signals through the skin and the skull in newborn mice that had undergone IUE at E10.5, E11.5 or E12.5. The EGFP signals were observed on the electroporated side of the cerebellum (Fig. 1B, left and middle panels), but when a series of pulses was sequentially applied in two diagonal directions, both sides of the cerebellum were transfected (Fig. 1B, right panel). More EGFP-positive cells were observed in mice that underwent IUE at E11.5 than at Carnitine palmitoyltransferase II E10.5 or E12.5 (Fig. 1B). EGFP was expressed in almost the entire half of the cerebellum that underwent

IUE at E11.5 (Fig. 1B). In contrast, EGFP expression was not observed in the middle of the vermis and the edge of the hemisphere of the cerebellum that underwent IUE at E10.5; EGFP signals were restricted in the middle of the vermis and the edge of the hemisphere when IUE was performed at E12.5 (Fig. 1B). Similarly, adenovirus vectors injected into the fourth ventricle at E10.5, E11.5 and E12.5 infect only the subpopulation of Purkinje cell progenitors that were born on the day of each injection (Hashimoto & Mikoshiba, 2003). Thus, it is likely that only cells that were located at the surface of the fourth ventricle at the time of IUE were transfected. To determine the cellular specificity of transfection, we fixed the cerebella at P14 and later and immunostained them for calbindin, a Purkinje cell marker. Again, more EGFP-positive cells were observed in the cerebellar sections taken from mice that underwent IUE at E11.5 than at E10.5 or E12.5 (Fig. 1C). The vast majority of EGFP-positive cells were immunopositive for calbindin in the cerebellum (Fig. 1C).

An EGFP-positive Purkinje cell whose soma was located at a distance more than one soma away from the Purkinje cell layer, defined by the rest of the EGFP-negative Purkinje cells, was counted as ‘mislocalized’. Statistical significance was defined by the χ2 test. For the statistical analysis of electrophysiological results, the Mann–Whitney U-test NVP-BKM120 datasheet was applied. Previous studies demonstrated that mouse Purkinje cells arise from the ventricular zone facing the fourth ventricle around E10–E13 (Miale & Sidman, 1961; Wang & Zoghbi, 2001; Hashimoto & Mikoshiba, 2003). Thus, to develop an IUE method for Purkinje cells, a plasmid

encoding EGFP under the control of the CAG promoter (CAG-EGFP) was injected selleck kinase inhibitor into the fourth ventricle of E10.5, E11.5 or E12.5 mice. To transfect Purkinje cell precursors, the electrodes were placed diagonally across the fourth ventricle with the anode above the cerebellar primordium at an angle of 90° or more to the targeted side of the upper rhombic lip (Fig. 1A

and Supporting Information, Fig. S1), and 33-V electrical pulses were applied five times (Fig. 1A). We observed bright EGFP signals through the skin and the skull in newborn mice that had undergone IUE at E10.5, E11.5 or E12.5. The EGFP signals were observed on the electroporated side of the cerebellum (Fig. 1B, left and middle panels), but when a series of pulses was sequentially applied in two diagonal directions, both sides of the cerebellum were transfected (Fig. 1B, right panel). More EGFP-positive cells were observed in mice that underwent IUE at E11.5 than at isothipendyl E10.5 or E12.5 (Fig. 1B). EGFP was expressed in almost the entire half of the cerebellum that underwent

IUE at E11.5 (Fig. 1B). In contrast, EGFP expression was not observed in the middle of the vermis and the edge of the hemisphere of the cerebellum that underwent IUE at E10.5; EGFP signals were restricted in the middle of the vermis and the edge of the hemisphere when IUE was performed at E12.5 (Fig. 1B). Similarly, adenovirus vectors injected into the fourth ventricle at E10.5, E11.5 and E12.5 infect only the subpopulation of Purkinje cell progenitors that were born on the day of each injection (Hashimoto & Mikoshiba, 2003). Thus, it is likely that only cells that were located at the surface of the fourth ventricle at the time of IUE were transfected. To determine the cellular specificity of transfection, we fixed the cerebella at P14 and later and immunostained them for calbindin, a Purkinje cell marker. Again, more EGFP-positive cells were observed in the cerebellar sections taken from mice that underwent IUE at E11.5 than at E10.5 or E12.5 (Fig. 1C). The vast majority of EGFP-positive cells were immunopositive for calbindin in the cerebellum (Fig. 1C).