Analysis of Gene Expression Using lacZ Reporter Mouse Lines.

Reporter mice transgenically expressing the bacterial (E. coli) lacZ gene encoding ß-galactosidase (ß-gal, EC 3.2.1.23) are a versatile and extensively used tool to study gene expression and cell lineage patterns. Enzymatic activity of the ß-gal reporter can be effectively visualized at cellular resolution either histochemically using 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) or by immunofluorescent detection using a ß-gal-specific antibody. Here, we summarize protocols for the localization of ß-gal expressing cells in whole embryos or organs as well as in histological tissue sections of lacZ reporter mice and discuss their limitations and common pitfalls.

CRISPR-Based Targeted Epigenetic Editing Enables Gene Expression Modulation of the Silenced Beta-Galactoside Alpha-2,6-Sialyltransferase 1 in CHO Cells.

Despite great efforts to control and modify gene expression of Chinese Hamster Ovary (CHO) cells by conventional genetic engineering approaches, i.e. overexpression or knockdown/-out, subclonal variation, induced unknown regulatory effects as well as overexpression stress are still a major hurdle for efficient cell line engineering and for unequivocal characterization of gene function. The use of epigenetic modulators - key players in CHO clonal heterogeneity - has only been marginally addressed so far. Here, we present the application of an alternative engineering strategy in CHO cells by utilizing targeted epigenetic editing tools that enable the turning-on or -off of genes without altering the genomic sequence. The present, but silent beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) gene is activated by targeting the catalytic domain (CD) of Ten-Eleven Translocation methylcytosine dioxygenase 1 (TET1) via deactivated Cas9 (dCas9) to its methylated promoter. Stable upregulation in up to 60% of transfected cells is achieved over a time span of more than 80 days. No difference in growth and recombinant protein productivity is observed between activated and control cultures. Re-silencing by targeted methylation via DNA methyltransferase (DNMT) 3A-CD resulted in an up to 5.4-fold reduction of ST6GAL1 mRNA expression in ST6GAL1 expressing cells. This proof-of-concept demonstrates the feasibility of using epigenetic editing tools to efficiently modulate gene expression and provide a promising complement to conventional genetic engineering in CHO cells.

The role of Hedgehog-responsive fibroblasts in facial nerve regeneration.

BACKGROUND: Facial nerve paralysis is a significant cause of morbidity, affecting facial appearance, emotional expression, speech, oral competence, and vision. A more complete understanding of the complex cellular events required for successful nerve regeneration may reveal new therapeutic targets. The role of fibroblasts in regeneration, and the process by which the nerve reforms its three-dimensional structure after a transection injury, are not fully understood. The Hedgehog signaling pathway has been shown to mediate nerve sheath formation during development. We therefore sought to characterize the role of Hedgehog-responsive cells following transection of the facial nerve. METHODS: Two transgenic mouse lines with reporters for the downstream effector of Hedgehog signaling, Gli1, were used. The animals underwent a unilateral facial nerve transection injury, and the contralateral side served as a control. Facial nerves were analyzed via immunohistochemistry and immunofluorescence at predetermined time points as the facial nerve regenerated after the transection injury. RESULTS: There was a statistically significant increase in Gli1+ cells both at the site of injury and within the distal nerve segment over time. Gli1+ cells are fibroblasts within the nerve and appear to contribute to the reformation of the nerve sheath after injury. CONCLUSION: These findings describe a key signaling pathway by which fibroblasts participate in motor nerve regeneration. Fibroblasts that reside within the nerve respond to injury and may represent a novel therapeutic target in the context of facial nerve regeneration after transection injury.

Effector Overlap between the lac and mel Operons of Escherichia coli: Induction of the mel Operon with ß-Galactosides.

The lac (lactose) operon (which processes ß-galactosides) and the mel (melibiose) operon (which processes α-galactosides) of Escherichia coli have a close historical connection. A number of shared substrates and effectors of the permeases and regulatory proteins have been reported over the years. Until now, ß-thiogalactosides like TMG (methyl-ß-d-thiogalactopyranoside) and IPTG (isopropyl-ß-d-thiogalactopyranoside) have not generally been considered to be inducers of the mel operon. The same is true for ß-galactosides such as lactose [ß-d-galactopyranosyl-(1→4)-d-glucose], which is a substrate but is not itself an inducer of the lac operon. This report shows that all three sugars can induce the mel operon significantly when they are accumulated in the cell by Lac permease. Strong induction by ß-thiogalactosides is observed in the presence of Lac permease, and strong induction by lactose (more than 200-fold) is observed in the absence of ß-galactosidase. This finding calls for reevaluation of TMG uptake experiments as assays for Lac permease that were performed with mel+ strains.IMPORTANCE The typical textbook picture of bacterial operons is that of stand-alone units of genetic information that perform, in a regulated manner, well-defined cellular functions. Less attention is given to the extensive interactions that can be found between operons. Well-described examples of such interactions are the effector molecules shared by the lac and mel operons. Here, we show that this set has to be extended to include ß-galactosides, which have been, until now, considered not to effect the expression of the mel operon. That they can be inducers of the mel operon as well as the lac operon has not been noted in decades of research because of the Escherichia coli genetic background used in previous studies.

Defects in neural guidepost structures and failure to remove leptomeningeal cells from the septal midline behind the interhemispheric fusion defects in Netrin1 deficient mice.

Corpus callosum (CC) is the largest commissural tract in mammalian brain and it acts to coordinate information between the two cerebral hemispheres. During brain development CC forms at the boundary area between the cortex and the septum and special transient neural and glial guidepost structures in this area are thought to be critical for CC formation. In addition, it is thought that the fusion of the two hemispheres in the septum area is a prerequisite for CC formation. However, very little is known of the molecular mechanisms behind the fusion of the two hemispheres. Netrin1 (NTN1) acts as an axon guidance molecule in the developing central nervous system and Ntn1 deficiency leads to the agenesis of CC in mouse. Here we have analyzed Ntn1 deficient mice to better understand the reasons behind the observed lack of CC. We show that Ntn1 deficiency leads to defects in neural, but not in glial guidepost structures that may contribute to the agenesis of CC. In addition, Nnt1 was expressed by the leptomeningeal cells bordering the two septal walls prior to fusion. Normally these cells are removed when the septal fusion occurs. At the same time, the Laminin containing basal lamina produced by the leptomeningeal cells is disrupted in the midline area to allow the cells to mix and the callosal axons to cross. In Ntn1 deficient embryos however, the leptomeninges and the basal lamina were not removed properly from the midline area and the septal fusion did not occur. Thus, NTN1 contributes to the formation of the CC by promoting the preceding removal of the midline leptomeningeal cells and interhemispheric fusion.

Timed conditional null of connexin26 in mice reveals temporary requirements of connexin26 in key cochlear developmental events before the onset of hearing.

Mutations in the Gjb2 gene, which encodes a gap junction protein connexin26 (Cx26), are the most prevalent form of hereditary deafness in humans and represent about half of non-syndromic congenital deafness cases in many ethnic populations. Cochlear potassium (K+) recycling in mature cochlea is required for normal hearing. It is thought that gap junctions are essential for K+ recycling and that Gjb2 mutations cause Gjb2-associated deafness by disrupting K+ recycling in mature cochlea. Here we present evidence showing that Gjb2 is required for normal development of the neonatal organ of Corti prior to the onset of the hearing in mice. In the conditional Gjb2 null (cCx26 null) mice, ribbon synapses in inner hair cells remained poorly developed, the afferent type I fibers failed to finish the refinement process to form convergent innervation to individual inner hair cells. The spontaneous depolarizing activities in the supporting cells, which normally diminish in the wild type cochleae after postnatal day 8 (P8), remained strong in the cochlea after P8 in the mutant mice. Furthermore, the deafness phenotype was readily generated only if the Cx26 expression in the organ of Corti was significantly reduced before P6. Similar amount of Cx26 reduction in more mature cochleae had a much weaker effect in damaging the hearing sensitivity. Our findings indicated that Cx26 plays essential roles in the maturation process of the organ of Corti prior to the establishment of high K+ in the endolymph and the onset of hearing. These results suggest that successful treatment of Cx26 deafness requires early intervention before the cochlea fully matures.

Stent-mediated gene delivery for site-specific transgene administration to the airway epithelium and management of tracheobronchial tumors.

BACKGROUND: Gene therapy is currently under investigation as a means of managing a variety of pulmonary diseases. Unfortunately, gene transfer to bronchial epithelium has been hampered by the lack of stable and efficient transduction. Recent studies have shown that gene vectors could be tethered to the metallic surfaces of intra-arterial stents. This approach enables efficacious and site-specific adenoviral gene delivery to the vascular endothelium. OBJECTIVES: We hypothesized that airway mesh stents impregnated with viral gene vectors could be used for local gene delivery to benign and malignant bronchial epithelium. METHODS: Serotype 5 adenoviral vectors (Ad5, E1-/E3-) containing the reporter genes green fluorescent protein (Ad.GFP) or ß-galactoside/LacZ (Ad.LacZ), or a therapeutic gene, Ad.INF-ß, were coupled to either metallic mesh disks or stents via anti-Ad knob antibodies. These platforms were assessed for their ability to transfect bronchial epithelial cells from both rats and humans, as well as murine (L1C2) and human (A549) lung cancer cell lines. Gene transfer was quantified by fluorescent microscopy, scanning fluorimetry for Ad.GFP, and light microscopy studies assessing ß-galactosidase staining for Ad.LacZ. Metallic mesh and stent-mediated gene transfer was also performed in a murine flank tumor model and in a rat endotracheal tumor model in order to evaluate the therapeutic potential. RESULTS: In these studies, murine and human non-small cell lung cancer (NSCLC) cells were successfully transfected with reporter genes in vitro. Ad.LacZ-complexed mesh successfully transfected reporter genes into established murine flank NSCLC tumors. In addition, Ad.LacZ-tethered stents could effectively transfect both tracheobronchial epithelium and submucosal glands in rats. Similar epithelial transfection was achieved in ex vivo human bronchial epithelium. Pilot in vivo experimentation provided data supporting the concept that therapeutic genes could also be delivered with this technology. In additional pilot in vivo experiments, the growth of murine flank tumors was inhibited by placement of mesh disks coupled with Ad.muINF-ß, and rats bearing endotracheal tumors demonstrated a trend towards prolonged survival with insertion of Ad.ratINF-ß-tethered stents. CONCLUSIONS: Stent-mediated gene delivery successfully enabled site-specific vector administration to target rat and human airway cells in cell culture, organ culture and in vivo. Local tracheobronchial gene delivery via stents could provide a viable clinical solution for overcoming the difficulties encountered with vector delivery within the lungs, in particular by lowering requisite vector titers and by directing desired vectors to areas of interest. This strategy may prove valuable for treating tumors involving the tracheobronchial tree, as well as other nonmalignant tracheobronchial disorders.

Phenotypic and genetic analysis of the German Malus Germplasm Collection in terms of type 1 and type 2 red-fleshed apples.

Red fruit flesh is a desirable trait in apple breeding, because red-fleshed apples are a novelty and therefore considered to be more attractive to consumers and contain more health beneficial compounds. The red fruit flesh coloration is based on an increased level of cyanidin 3-galactoside, an anthocyanin whose biosynthesis is regulated by the MYB-type transcription factors MdMYB10 or MdMYB110a, respectively. A repeated segment in the MdMYB10 promoter allele R6 results in a gain-of-function mutation visible as red pigmentation of fruit skin and flesh and all vegetative tissues. Red-fleshed apple genotypes containing this R6 allele belong to the type 1 red-fleshed apple, which is known to be linked to some negative traits like astringent taste and internal flesh browning disorder. In type 2 red-fleshed apples the fruit flesh coloration is not inevitably linked with skin and leaf color. This red-fleshed apple phenotype, which is a result of increased expression of MdMYB110a, seems to be more useful for breeding, but it can be found rather seldom. In the present study 357 Malus accessions of the German Malus Germplasm Collection were evaluated for red fruit flesh coloration and the presence of the MdMYB10 R1 (not mutated) and R6 promoter alleles. Among them a total of 40 accessions were identified which contain the R6 allele. 37 accessions showed a red coloration of the fruit flesh. All these accessions belong to type 1 red-fleshed apple. No type 2 red-fleshed apple could be found. Three accessions with R6 allele had non-red-fleshed apples. 312 other non-red-fleshed accessions contained only the R1 allele. Five non-red-fleshed accessions contained a new promoter allele with an unexpected size of ~1 kbp. Sequencing of this allele detected the insertion of a non-autonomous apple transposon.

Specificity and efficiency of reporter expression in adult neural progenitors vary substantially among nestin-CreER(T2) lines.

Transgenic lines expressing a controllable form of Cre recombinase have become valuable tools for manipulating gene expression in adult neural progenitors and their progeny. Neural progenitors express several proteins that distinguish them from mature neurons, and the promoters for these genes have been co-opted to produce selective transgene expression within this population. To date, nine CreER(T2) transgenic lines have been designed using the nestin promoter; however, only a subset are capable of eliciting expression within both neurogenic zones of the adult brain. Here we compare three such nestin-CreER(T2) lines to evaluate specificity of expression and efficiency of recombination. Each line was examined by using three different Cre reporter strains that varied in sensitivity. We found that all three nestin-CreER(T2) strains induced reporter expression within the main neurogenic areas, albeit to varying degrees depending on the reporter. Unexpectedly, we found that two of the three lines induced substantial reporter expression outside of neurogenic areas. These lines produced strong labeling in cerebellar granule neurons, with additional expression in the cortex, hippocampus, striatum, and thalamus. Reporter expression in the third nestin-CreER(T2) line was considerably more specific, but was also less efficient, labeling a smaller percentage of the target population than the other two drivers. Our findings suggest that each nestin-CreER(T2) line may best serve different experimental needs, depending on whether specificity or efficiency is of greatest concern. Our study further demonstrates that each new pair of driver and responder lines should be evaluated independently, as both components can significantly influence the resulting expression pattern.

Ras inhibition boosts galectin-7 at the expense of galectin-1 to sensitize cells to apoptosis.

Galectins are a family of ß-galactoside-binding lectins that exert diverse extracellular and intracellular effects. Galectin-7 and galectin-1 show opposing effects on proliferation and survival in different cell types. Galectin-7 is a p53-induced gene and an enhancer of apoptosis, whereas galectin-1 induces tumorigenicity and resistance to apoptosis in several types of cancers. We show here that in cells derived from neurofibromin-deficient (Nf1(-/-)) malignant peripheral nerve sheath tumors (MPNSTs), Ras inhibition by S-trans,trans-farnesylthiosalicylic-acid (FTS; Salirasib) shifts the pattern of galectin expression. Whereas FTS decreased levels of both active Ras and galectin-1 expression, it dramatically increased both the mRNA and protein expression levels of galectin-7. Galectin-7 accumulation was mediated through JNK inhibition presumably resulting from the observed induction of p53, and was negatively regulated by the AP-1 inhibitor JDP2. Expression of galectin-7 by itself decreased Ras activation in ST88-14 cells and rendered them sensitive to apoptosis. This observed shift in galectin expression pattern together with the accompanying shift from cell proliferation to apoptosis represents a novel pattern of Ras inhibition by FTS. This seems likely to be an important phenomenon in view of the fact that both enhanced cell proliferation and defects of apoptosis constitute major hallmarks of human cancers and play a central role in the resistance of MPNSTs to anti-cancer treatments. These findings suggest that FTS, alone or in combination with chemotherapy agents, may be worth developing as a possible treatment for MPNSTs.

A cloning vector employing a versatile ß-glucosidase as an indicator for recombinant clones.

A mutant glucosidase, cpGluT, with activity toward chromogenic substrates (X-gal [5-bromo-4-chloro-3-idolyl-ß-d-galactoside] and indican) and a fluorogenic 4-methylumbeliferyl-ß-d-glucopyranoside (MUG) was constructed by replacing the monomeric ß-glucosidase region (E314-N326) with designed multiple cloning sites. When expressed in hosts (lacZ+ and lacZ-), a vector containing the cpGluT produced a colored or fluorescent phenotype according to the substrate supplemented on LB plates without any inducer. cpGluT is readily incorporable into customized vectors and does not require special hosts to detect recombinant plasmids, thereby making screening recombinants more effective and less expensive.

The DC2.3 gene in Caenorhabditis elegans encodes a galectin that recognizes the galactoseß1→4fucose disaccharide unit.

Galectins comprise a large family of ß-galactoside-binding proteins in animals and fungi. We previously isolated cDNAs of 10 galectin and galectin-like genes (lec-1 to lec-6 and lec-8 to lec-11) from Caenorhabditis elegans and characterized the carbohydrate-binding properties of their recombinant proteins. In the present study, we isolated cDNA corresponding to an open reading frame of the DC2.3a gene from C. elegans total RNA; this cDNA encodes another potential galectin. A recombinant DC2.3a protein was expressed in Escherichia coli and used for analysis. The protein displayed hemagglutinating activity against rabbit erythrocytes, bound to an asialofetuin-Sepharose column, and was eluted with lactose. Furthermore, frontal affinity chromatography (FAC) analysis confirmed that DC2.3a recognized oligosaccharides with a non-reducing terminal galactose. According to these results, we designated DC2.3 as lec-12. The carbohydrate-binding property of the recombinant DC2.3a/LEC-12a was essentially similar to that of LEC-6. Additionally, DC2.3a/LEC-12a and LEC-6 showed higher affinities for the galactoseß1→4fucose (Galß1→4Fuc) disaccharide than for N-acetyllactosamine. This suggests that the principal recognition unit is the Galß1→4Fuc disaccharide as in the case of the C. elegans galectins. However, the recombinant DC2.3a/LEC-12a showed weak affinity for N-glycan E3, which was previously shown to be a preferential endogenous ligand for LEC-6. The DC2.3a/LEC-12a endogenous ligand structures appear to be somewhat different but contain the same galactose-fucose recognition motif.

Precerebellar cell groups in the hindbrain of the mouse defined by retrograde tracing and correlated with cumulative Wnt1-cre genetic labeling.

The precerebellar nuclei are hindbrain and spinal cord centers that send fibers to the cerebellum. The neurons of the major hindbrain precerebellar nuclei are derived from the rhombic lip. Wnt1, a developmentally important gene involved in intercellular signaling, is expressed in the developing rhombic lip. We sought to investigate the relationship between the cell clusters expressing Wnt1 and the precerebellar nuclei in the hindbrain. We therefore defined the hindbrain precerebellar nuclei by retrograde tracing, following cerebellar injections of HRP, and compared these results with the cell clusters expressing Wnt1 in newborn mice. We found that 39 distinct hindbrain nuclei project to the cerebellum. Of these nuclei, all but three (namely the oral pontine reticular nucleus, the caudal pontine reticular nucleus, and the subcoeruleus nucleus) contain neurons expressing Wnt1. This shows a high degree of overlap between the precerebellar nuclei and the nuclei that express Wnt1. However, it should be noted that neurons expressing Wnt1 are also found in the superior olivary complex, which is a basal plate derivative lacking cerebellar projections.

Transplantation of a mammary stromal cell line into a mammary fat pad: development of the site-specific in vivo analysis system for mammary stromal cells.

The interaction between mammary epithelial and stromal tissue is considered to be important in breast tissue development. In this study, we developed a transplantation procedure for the mammary stromal fibroblastic cell line (MSF) to examine its life in vivo. First we established MSF cells which stably expressed lacZ (lacZ/MSF) and had characteristics of mammary stromal cells. The lacZ/MSF cells were then transplanted into a cleared mammary fat pad of syngenic mice with and without mammary primary epithelial organoids. Whole mount X-gal and carmine staining of the transplants revealed that a number of undifferentiated lacZ/MSF cells survived around the mammary epithelial tissue when transplanted with organoids. These results indicate that transplantation of MSF cells into mammary fat pad was accomplished by co-transplantation with primary mammary organoids. Finally, we discuss the application of transplantation procedure for in vivo studies of the mammary stromal tissue development and stromal-epithelial interactions.

LEUNIG_HOMOLOG and LEUNIG regulate seed mucilage extrusion in Arabidopsis.

LEUNIG (LUG) and LEUNIG_HOMOLOG (LUH) encode two closely related Arabidopsis proteins, belonging to the Gro/TLE family of transcriptional co-repressors. These two genes were previously shown to exhibit partially overlapping functions in embryo and flower development. In this report, the role of both LUH and LUG on seed mucilage extrusion was examined. Seed mucilage extrusion occurs after the seeds are imbibed, serving as functional aid in seed hydration, germination, and dispersal. While luh-1 mutants exhibited strong defects in seed mucilage extrusion, lug-3 mutants exhibited a minor phenotype in mucilage extrusion. Further characterization indicates that luh-1 does not exhibit any obvious defect in seed epidermal cell differentiation, mucilage synthesis, or mucilage deposition, suggesting a specific role of LUH in mucilage extrusion. This seed mucilage phenotype of luh-1 is identical to that of mucilage modified 2 (mum2) mutants. MUM2 encodes a ß-galactosidase required for the modification of the mucilage. Quantitative reverse transcription polymerase chain reaction of RNA extracted from siliques detected a slight decrease of MUM2 mRNA in the luh-1 mutant compared to the wild type. Together, LUH and possibly LUG may specifically regulate mucilage extrusion by promoting the expression of genes required for mucilage maturation.

Discovery and structural characterization of a novel glycosidase family of marine origin.

The genomic data on heterotrophic marine bacteria suggest the crucial role that microbes play in the global carbon cycle. However, the massive presence of hypothetical proteins hampers our understanding of the mechanisms by which this carbon cycle is carried out. Moreover, genomic data from marine microorganisms are essentially annotated in the light of the biochemical knowledge accumulated on bacteria and fungi which decompose terrestrial plants. However marine algal polysaccharides clearly differ from their terrestrial counterparts, and their associated enzymes usually constitute novel protein families. In this study, we have applied a combination of bioinformatics, targeted activity screening and structural biology to characterize a hypothetical protein from the marine bacterium Zobellia galactanivorans, which is distantly related to GH43 family. This protein is in fact a 1,3-α-3,6-anhydro-l-galactosidase (AhgA) which catalyses the last step in the degradation pathway of agars, a family of polysaccharides unique to red macroalgae. AhgA adopts a ß-propeller fold and displays a zinc-dependent catalytic machinery. This enzyme is the first representative of a new family of glycoside hydrolases, especially abundant in coastal waters. Such genes of marine origin have been transferred to symbiotic microbes associated with marine fishes, but also with some specific human populations.

Mutational tuning of galectin-3 specificity and biological function.

Galectins are defined by a conserved ß-galactoside binding site that has been linked to many of their important functions in e.g. cell adhesion, signaling, and intracellular trafficking. Weak adjacent sites may enhance or decrease affinity for natural ß-galactoside-containing glycoconjugates, but little is known about the biological role of this modulation of affinity (fine specificity). We have now produced 10 mutants of human galectin-3, with changes in these adjacent sites that have altered carbohydrate-binding fine specificity but that retain the basic ß-galactoside binding activity as shown by glycan-array binding and a solution-based fluorescence anisotropy assay. Each mutant was also tested in two biological assays to provide a correlation between fine specificity and function. Galectin-3 R186S, which has selectively lost affinity for LacNAc, a disaccharide moiety commonly found on glycoprotein glycans, has lost the ability to activate neutrophil leukocytes and intracellular targeting into vesicles. K176L has increased affinity for ß-galactosides substituted with GlcNAcß1-3, as found in poly-N-acetyllactosaminoglycans, and increased potency to activate neutrophil leukocytes even though it has lost other aspects of galectin-3 fine specificity. G182A has altered carbohydrate-binding fine specificity and altered intracellular targeting into vesicles, a possible link to the intracellular galectin-3-mediated anti-apoptotic effect known to be lost by this mutant. Finally, the mutants have helped to define the differences in fine specificity shown by Xenopus, mouse, and human galectin-3 and, as such, the evidence for adaptive change during evolution.

Therapeutic time window of mesenchymal stem cells derived from bone marrow after cerebral ischemia.

Intravenous transplantation of mesenchymal stem cells (MSCs) prepared from bone marrow ameliorates functional deficits in rat cerebral infarction models. Although several hypotheses to account for these therapeutic effects have been suggested, angiogenesis is thought to be largely responsible. MSCs were intravenously infused into rats in the relatively later time points after permanent middle cerebral artery occlusion (MCAO) to determine if they could contribute to neovascularization and functional recovery. Although MRI analysis revealed that only rats receiving MSCs 7days after MCAO exhibited decreased ischemic lesion volume than the control group, all MSCs treated rats receiving MSCs up to 1month after MCAO had greater angiogenesis near the border of the ischemic lesions and showed greater functional recovery in the treadmill stress test than did control rats. Thus, these results suggest that the time window of MSC therapy is at least over 1 month after cerebral infarction in the rat permanent MCAO model, and systemic delivery of MSCs in the later phase after cerebral ischemia may have beneficial effect through an angiogenic mechanism.

Establishment of a simple and rapid method to screen for strong promoters in Bacillus subtilis.

Using published plasmid vectors containing the bgaB gene encoding a heat-stable beta-galactosidase, we have been unable to fuse strong promoters to this reporter gene. In addition, we could not analyze the promoter strength by a plate assay. Therefore, we constructed an Escherichia coli-Bacillus subtilis shuttle vector to allow the cloning of strong promoters and their rapid analysis in B. subtilis by plating on X-Gal plates in the presence of the inducer IPTG. We show that the blue color of the colonies reflects the strength of the promoters, which was verified by measuring the beta-galactosidase activities.

Radiation damage increases Purkinje neuron heterokaryons in neonatal cerebellum.

OBJECTIVE: Recent studies have shown that in radiated and bone marrow transplanted mice, bone marrow-derived cells (BMDCs) fuse with Purkinje neurons resulting in the formation of binucleated heterokaryons. Here we investigated whether radiation plays a role in the formation of Purkinje neuron heterokaryons. METHODS: Fused cells were identified by reporter gene expression in mice, carrying floxed LacZ (R26R-LacZ) in all cells and Cre in hematopoietic-derived cells. Cell fusion was confirmed by the presence of two nuclei. The number of fused Purkinje neurons was studied in: 1) whole-body radiated newborn and adult R26R-LacZ mice, transplanted with bone marrow cells expressing Cre; 2) in newborn and adult mice that received different doses of radiation to the head; and 3) in radiated and non-radiated newborns treated with a myeloablative drug before bone marrow transplantation. RESULTS: In neonatal, but not in adult cerebelleum, radiation-in a dose-dependent manner-induces a dramatic increase in the number of fused Purkinje neurons. INTERPRETATION: Increase recruitment of BMDCs into the cerebellum, radiation damage to cerebellar cells, or both, increase the formation of fused Purkinje cells. BMDC-Purkinje heterokaryons formation may reflect an endogeneous neuronal repair mechanism, or it could be a by-product of radiation-induced inflammation. In either case, fused Purkinje neurons increase following radiation damage in the developing cerebellum. The above observations reveal a novel consequence of head radiation in neonatal rodents. It will be interesting to determine if similar increase in the number of binucleated Purkinje neurons, occurs in children that receive radiation during early development. Ann Neurol 2009;66:100-109.