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Summary Efforts to overcome resistance to immune checkpoint blockade therapy have focused on vaccination strategies using neoepitopes, although they cannot be applied on a large scale due to the “private” nature of cancer mutations. Here, we show that infection of tumor cells with Salmonella induces the opening of membrane hemichannels and the extracellular release of proteasome-generated peptides by the exacerbation of endoplasmic reticulum (ER) stress. Peptides released by cancer cells foster an antitumor response in vivo, both in mice bearing B16F10 melanomas and in dogs suffering from osteosarcoma. Mass spectrometry analysis on the supernatant of human melanoma cells revealed 12 peptides capable of priming healthy-donor CD8+ T cells that recognize and kill human melanoma cells in vitro and when xenotransplanted in vivo. Hence, we identified a class of shared tumor antigens that are generated in ER-stressed cells, such as tumor cells, that do not induce tolerance and are not presented by healthy cells., Graphical abstract, Highlights • Tumor cell infection with Salmonella induces the release of immunogenic peptides • Peptide-based vaccine boosts a strong antitumor response in dog osteosarcoma • MS analysis identified shared peptides released by human melanoma cell lines • Twelve identified peptides are capable of inducing a tumor-specific CD8 response, Melacarne et al. demonstrate that Salmonella exacerbates endoplasmic reticulum stress in tumor cells, which induces hemichannel opening and the subsequent release of peptides in the extracellular milieu. Released peptides are immunogenic, shared among tumors, and can be exploited as an anticancer vaccine to prevent metastasis occurrence.

Background Animal studies show that high fat (HF) diet-induced gut microbiota contributes to the development of obesity. Oil composition of high-fat diet affects metabolic inflammation differently with deleterious effects by saturated fat. The aim of the present study was to examine the diversity and metabolic capacity of the cecal bacterial community in C57BL/6 N mice administered two different diets, enriched respectively with coconut oil (HFC, high in saturated fat) or soy oil (HFS, high in polyunsaturated fat). The relative impact of each hypercaloric diet was evaluated after 2 and 8 weeks of feeding, and compared with that of a low-fat, control diet (LF). Results The HFC diet induced the same body weight gain and fat storage as the HFS diet, but produced higher plasma cholesterol levels after 8 weeks of treatment. At the same time point, the cecal microbiota of HFC diet-fed mice was characterized by an increased relative abundance of Allobaculum, Anaerofustis, F16, Lactobacillus reuteri and Deltaproteobacteria, and a decreased relative abundance of Akkermansia muciniphila compared to HFS mice. Comparison of cecal microbiota of high-fat fed mice versus control mice indicated major changes that were shared between the HFC and the HFS diet, including the increase in Lactobacillus plantarum, Lutispora, and Syntrophomonas, while some other shifts were specifically associated to either coconut or soy oil. Prediction of bacterial gene functions showed that the cecal microbiota of HFC mice was depleted of pathways involved in fatty acid metabolism, amino acid metabolism, xenobiotic degradation and metabolism of terpenoids and polyketides compared to mice on HFS diet. Correlation analysis revealed remarkable relationships between compositional changes in the cecal microbiota and alterations in the metabolic and transcriptomic phenotypes of high-fat fed mice. Conclusions The study highlights significant differences in cecal microbiota composition and predictive functions of mice consuming a diet enriched in coconut vs soy oil. The correlations established between specific bacterial taxa and various traits linked to host lipid metabolism and energy storage give insights into the role and functioning of the gut microbiota that may contribute to diet-induced metabolic disorders. Electronic supplementary material The online version of this article (10.1186/s12864-018-5202-z) contains supplementary material, which is available to authorized users.

CRISPR-Cas9 technology is a relatively recently developed tool for easy and efficient targeting of DNA. However, its efficiency for the repair of a mutated sequence is low. Moreover, most CRISPR-based gene correction approaches require the use of an exogenous template. Here, we investigated whether we could use the CRISPR-Cas9 system and the autologous repair machinery to correct human recessive genetic disorders having two different mutations in two alleles (compound heterozygotes). We reasoned that by targeting an intronic sequence located between the two mutations, we could generate at least one normal allele via the repair of induced double-strand breaks through either gene conversion or mitotic crossover. In particular, using a simple hypoxanthine-guanine phosphoribosyltransferase (

Metastasis is facilitated by the formation of a "premetastatic niche," which is fostered by primary tumor-derived factors. Colorectal cancer (CRC) metastasizes mainly to the liver. We show that the premetastatic niche in the liver is induced by bacteria dissemination from primary CRC. We report that tumor-resident bacteria Escherichia coli disrupt the gut vascular barrier (GVB), an anatomical structure controlling bacterial dissemination along the gut-liver axis, depending on the virulence regulator VirF. Upon GVB impairment, bacteria disseminate to the liver, boost the formation of a premetastatic niche, and favor the recruitment of metastatic cells. In training and validation cohorts of CRC patients, we find that the increased levels of PV-1, a marker of impaired GVB, is associated with liver bacteria dissemination and metachronous distant metastases. Thus, PV-1 is a prognostic marker for CRC distant recurrence and vascular impairment, leading to liver metastases. Copyright © 2021 Elsevier Inc. All rights reserved.

Controlling oxidative stress through the activation of antioxidant pathways is crucial in bone homeostasis, and impairments of the cellular defense systems involved contribute to the pathogenesis of common skeletal diseases. In this work we focused on the dipeptidyl peptidase 3 (DPP3), a poorly investigated ubiquitous zinc-dependent exopeptidase activating the Keap1-Nrf2 antioxidant pathway. We showed Dpp3 expression in bone and, to understand its role in this compartment, we generated a Dpp3 knockout (KO) mouse model and specifically investigated the skeletal phenotype. Adult Dpp3 KO mice showed a mild growth defect, a significant increase in bone marrow cellularity, and bone loss mainly caused by increased osteoclast activity. Overall, in the mouse model, lack of DPP3 resulted in sustained oxidative stress and in alterations of bone microenvironment favoring the osteoclast compared to the osteoblast lineage. Accordingly, in vitro studies revealed that Dpp3 KO osteoclasts had an inherent increased resorptive activity and ROS production, which on the other hand made them prone to apoptosis. Moreover, absence of DPP3 augmented bone loss after estrogen withdrawal in female mice, further supporting its relevance in the framework of bone pathophysiology. Overall, we show a nonredundant role for DPP3 in the maintenance of bone homeostasis and propose that DPP3 might represent a possible new osteoimmunological player and a marker of human bone loss pathology. © 2019 American Society for Bone and Mineral Research.

There is wide agreement that cell fusion is a physiological process in cells in mammalian bone, muscle and placenta. In other organs, such as the cerebellum, cell fusion is controversial. The liver contains a considerable number of polyploid cells: They are commonly believed to originate by genome endoreplication, although the contribution of cell fusion to polyploidization has not been excluded. Here, we address the topic of cell fusion in the liver from a historical point of view. We discuss experimental evidence clearly supporting the hypothesis that cell fusion occurs in the liver, specifically when bone marrow cells were injected into mice and shown to rescue genetic hepatic degenerative defects. Those experiments-carried out in the latter half of the last century-were initially interpreted to show "transdifferentiation", but are now believed to demonstrate fusion between donor macrophages and host hepatocytes, raising the possibility that physiologically polyploid cells, such as hepatocytes, could originate, at least partially, through homotypic cell fusion. In support of the homotypic cell fusion hypothesis, we present new data generated using a chimera-based model, a much simpler model than those previously used. Cell fusion as a road to polyploidization in the liver has not been extensively investigated, and its contribution to a variety of conditions, such as viral infections, carcinogenesis and aging, remains unclear.

Influenza A virus is the principal agent responsible of the respiratory tract's infections in humans. Every year, highly pathogenic and infectious strains with new antigenic assets appear, making ineffective vaccines so far developed. The discovery of RNA interference (RNAi) opened the way to the progress of new promising drugs against Influenza A virus and also to the introduction of disease resistance traits in genetically modified animals. In this paper, we show that Madin-Darby Canine Kidney (MDCK) cell line expressing short hairpin RNAs (shRNAs) cassette, designed on a specific conserved region of the nucleoprotein (NP) viral genome, can strongly inhibit the viral replication of four viral strains sharing the target sequence, reducing the viral mRNA respectively to 2.5×10(-4), 7.5×10(-5), 1.7×10(-3), 1.9×10(-4) compared to the control, as assessed by real-time PCR. Moreover, we demonstrate that during the challenge with a viral strain bearing a single mismatch on the target sequence, although a weaker inhibition is observed, viral mRNA is still lowered down to 1.2×10(-3) folds in the shRNA-expressing clone compared to the control, indicating a broad potential use of this approach. In addition, we developed a highly predictive and fast screening test of siRNA sequences based on dual-luciferase assay, useful for the in vitro prediction of the potential effect of viral inhibition. In conclusion, these findings reveal new siRNA sequences able to inhibit Influenza A virus replication and provide a basis for the development of siRNAs as prophylaxis and therapy for influenza infection both in humans and animals.

Background The association between maternal infection and neurodevelopmental defects in progeny is well established, although the biological mechanisms and the pathogenic trajectories involved have not been defined. Methods Pregnant dams were injected intraperitoneally at gestational day 9 with polyinosinic:polycytidylic acid. Neuronal development was assessed by means of electrophysiological, optical, and biochemical analyses. Results Prenatal exposure to polyinosinic:polycytidylic acid causes an imbalanced expression of the Na+-K+-2Cl− cotransporter 1 and the K+-Cl− cotransporter 2 (KCC2). This results in delayed gamma-aminobutyric acid switch and higher susceptibility to seizures, which endures up to adulthood. Chromatin immunoprecipitation experiments reveal increased binding of the repressor factor RE1-silencing transcription (also known as neuron-restrictive silencer factor) to position 509 of the KCC2 promoter that leads to downregulation of KCC2 transcription in prenatally exposed offspring. Interleukin-1 receptor type I knockout mice, which display braked immune response and no brain cytokine elevation upon maternal immune activation, do not display KCC2/Na+-K+-2Cl− cotransporter 1 imbalance when implanted in a wild-type dam and prenatally exposed. Notably, pretreatment of pregnant dams with magnesium sulfate is sufficient to prevent the early inflammatory state and the delay in excitatory-to-inhibitory switch associated to maternal immune activation. Conclusions We provide evidence that maternal immune activation hits a key neurodevelopmental process, the excitatory-to-inhibitory gamma-aminobutyric acid switch; defects in this switch have been unequivocally linked to diseases such as autism spectrum disorder or epilepsy. These data open the avenue for a safe pharmacological treatment that may prevent the neurodevelopmental defects caused by prenatal immune activation in a specific pregnancy time window.

At weaning, piglets are exposed to many stressors, such as separation from the sow, mixing with other litters, end of lactational immunity, and a change in their environment and gut microbiota. The sudden change of feeding regime after weaning causes morphological and histological changes in the small intestine which are critical for the immature digestive system. Sixteen female piglets were studied to assess the effect of sorbic acid supplementation on the small intestine tissue transcriptome. At weaning day (T0, piglet age 28 days), four piglets were sacrificed and ileal tissue samples collected. The remaining 12 piglets were weighed and randomly assigned to different postweaning (T5, piglet age 33 days) diets. Diet A (n = 6) contained 5 g/kg of sorbic acid. In diet B (n = 6), the organic acids were replaced by barley flour. Total RNA was isolated and then hybridized to CombiMatrix CustomArray™ 90-K platform microarrays, screening about 30 K genes. Even though diet had no detectable effect on the transcriptome during the first 5 days after weaning, results highlighted some of the response mechanisms to the stress of weaning occurring in the piglet gut. A total of 205 differentially expressed genes were used for functional analysis using the bioinformatics tools BLAST2GO, Ingenuity Pathway Analysis 8.0, and Dynamic Impact Approach (DIA). Bioinformatic analysis revealed that apoptosis, RIG-I-like, and NOD-like receptor signaling were altered as a result of weaning. Interferons and caspases gene families were the most activated after weaning in response to piglets to multiple stressors. Results suggest that immune and inflammatory responses were activated and likely are a cause of small intestine atrophy as revealed by a decrease in villus height and villus/crypt ratio.

Mice fed long-term high-fat diets (HFD) are an established model for human metabolic disorders, such as obesity and diabetes. However, also the effects of short-term HFD feeding should be investigated to understand which are the first events that trigger the onset of a pre-disease condition, the so-called metabolic syndrome, that increases the risk of developing clinical diseases. In this study, C57BL/6N mice were fed a control diet (CTR) or a HFD for 1 (T1) or 2 weeks (T2). Metabolic and histological effects were examined. Cecum transcriptomes of HFD and CTR mice were compared at T2 by microarray analysis. Differentially expressed genes were validated by real-time PCR in the cecum and in the liver. After 2 weeks of diet administration, HFD mice showed an altered expression pattern in only seven genes, four of which are involved in the circadian clock regulatory pathway. Real-time PCR confirmed microarray results of the cecum and revealed the same trend of clock gene expression changes in the liver. These findings suggest that clock genes may play an important role in early controlling gut output systems in response to HFD in mice and that their expression change may also represent an early signaling of the development of an intestinal pro-inflammatory status.

// Michela Lizier 1,2,3 , Achille Anselmo 2 , Stefano Mantero 1,2 , Francesca Ficara 1,2 , Marianna Paulis 1,2 , Paolo Vezzoni 1,2 , Franco Lucchini 3 and Giovanni Pacchiana 1,2 1 Milan Unit, Istituto di Ricerca Genetica e Biomedica, CNR, Milan,Italy 2 Humanitas Clinical and Research Center, Rozzano, Milan, Italy 3 Centro Ricerche Biotecnologiche, Universita Cattolica del Sacro Cuore, Cremona, Milan, Italy Correspondence to: Franco Lucchini, email: // Keywords : neu oncogene; cell fusion; metastatic spread; macrophage; reporter genes Received : April 20, 2016 Accepted : August 11, 2016 Published : August 22, 2016 Abstract Cell fusion between neoplastic and normal cells has been suggested to play a role in the acquisition of a malignant phenotype. Several studies have pointed to the macrophage as the normal partner in this fusion, suggesting that the fused cells could acquire new invasive properties and become able to disseminate to distant organs. However, this conclusion is mainly based on studies with transplantable cell lines. We tested the occurrence of cell fusion in the MMTV-neu model of mouse mammary carcinoma. In the first approach, we generated aggregation chimeras between GFP/neu and RFP/neu embryos. Tumor cells would display both fluorescent proteins only if cell fusion with normal cells occurred. In addition, if cell fusion conferred a growth/dissemination advantage, cells with both markers should be detectable in lung metastases at increased frequency. We confirmed that fused cells are present at low but consistent levels in primary neoplasms and that the macrophage is the normal partner in the fusion events. Similar results were obtained using a second approach in which bone marrow from mice carrying the Cre transgene was transplanted into MMTV-neu/LoxP-tdTomato transgenic animals, in which the Tomato gene is activated only in the presence of CRE recombinase. However, no fused cells were detected in lung metastases in either model. We conclude that fusion between macrophages and tumor cells does not confer a selective advantage in our spontaneous model of breast cancer, although these data do not rule out a possible role in models in which an inflammation environment is prominent.

The synthesis of heterologous proteins in lactobacilli is strongly influenced by the promoter selected for the expression. In addition, the activity of the promoters themselves may vary among different bacterial hosts. Three different promoters were investigated for their capability to drive enhanced green fluorescent protein (EGFP) expression in Lactococcus lactis spp. cremoris MG1363, in Lactobacillus reuteri DSM 20016(T) and in five L. reuteri strains isolated from chicken crops. The promoters of the Lactobacillus acidophilus surface layer protein gene (slp), L. acidophilus lactate dehydrogenase gene (ldhL) and enterococcal rRNA adenine N-6-methyltransferase gene (ermB) were fused to the coding sequence of EGFP and inserted into the backbone of the pTRKH3 shuttle vector (pTRKH3-slpGFP, pTRKH3-ldhGFP, pTRKH3-ermGFP). Besides conventional analytical methods, a new quick fluorimetric approach was set up to quantify the EGFP fluorescence in transformed clones using the Qubit() fluorometer. ermB proved to be the most effective promoter in L. reuteri isolates, producing 3.90 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Under the same conditions, the ldhL promoter produced 2.66 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Even though the slp promoter was efficient in L. lactis spp. cremoris MG1363, it was nearly inactive both in L. reuteri DSM 20016(T) and in L. reuteri isolates.

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated 9 (Cas9) technology has been recently added to the tools allowing efficient and easy DNA targeting, representing a very promising approach to gene engineering. Using the CRISPR/Cas9 system we have driven the integration of exogenous DNA sequences to the X-linked Hprt gene of mouse embryonic stem cells. We show here that a simple fluorescence in situ hybridization (FISH)-based strategy allows the detection and the frequency evaluation of non-specific integrations of a given plasmid. FISH analysis revealed that these integrations do not match the software predicted off-target loci. We conclude that the frequency of these CRISPR-mediated off-target DNA cuts is negligible, since, due to the occurrence of spontaneous double-strand breaks, we observed more aspecific plasmid integrations than those corresponding to predicted off-target sites.

// Marianna Paulis 1,2 , Alessandra Castelli 1,2 , Lucia Susani 1,2 , Michela Lizier 2,3 , Irina Lagutina 4 , Maria Luisa Focarelli 1,2 , Camilla Recordati 5 , Paolo Uva 6 , Francesca Faggioli 1,2 , Tui Neri 1,2 , Eugenio Scanziani 5,7 , Cesare Galli 4,8 , Franco Lucchini 3 , Anna Villa 1,2,* and Paolo Vezzoni 1,2,* 1 Milan Unit, Istituto di Ricerca Genetica e Biomedica, CNR, Milan, Italy 2 Humanitas Clinical and Research Center, Rozzano, Milan, Italy 3 Centro Ricerche Biotecnologiche, Universita Cattolica del Sacro Cuore, Cremona, Italy 4 Avantea, Cremona, Italy 5 Mouse and Animal Pathology Laboratory, Fondazione Filarete, Milan, Italy 6 CRS4 Bioinformatics Laboratory, Parco Scientifico e Tecnologico POLARIS, Pula, Cagliari, Italy 7 Department of Veterinary Sciences and Public Health, University of Milan, Milan, Italy 8 Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia, Bologna, Italy * These authors jointly supervised this work Correspondence to: Paolo Vezzoni, email: // Marianna Paulis, email: // Keywords : embryonic stem cells, chromosome transplantation, microcell fusion, genomic disorders, cell therapy Received : September 28, 2015 Accepted : October 01, 2015 Published : October 17, 2015 Abstract Genomic disorders resulting from large rearrangements of the genome remain an important unsolved issue in gene therapy. Chromosome transplantation, defined as the perfect replacement of an endogenous chromosome with a homologous one, has the potential of curing this kind of disorders. Here we report the first successful case of chromosome transplantation by replacement of an endogenous X chromosome carrying a mutation in the Hprt genewith a normal one in mouse embryonic stem cells (ESCs), correcting the genetic defect. The defect was also corrected by replacing the Y chromosome with an X chromosome. Chromosome transplanted clones maintained in vitro and in vivo features of stemness and contributed to chimera formation. Genome integrity was confirmed by cytogenetic and molecular genome analysis. The approach here proposed, with some modifications, might be used to cure various disorders due to other X chromosome aberrations in induced pluripotent stem (iPS) cells derived from affected patients.

The gut microbiota has been shown to be involved in host energy homeostasis and diet-induced metabolic disorders. To gain insight into the relationships among diet, microbiota and the host, we evaluated the effects of a high-fat (HF) diet on the gut bacterial community in weaning mice. C57BL/6 mice were fed either a control diet or a diet enriched with soy oil for 1 and 2 weeks. Administration of the HF diet caused an increase in plasma total cholesterol levels, while no significant differences in body weight gain were observed between the two diets. Denaturing gradient gel electrophoresis (DGGE) profiles indicated considerable variations in the caecal microbial communities of mice on the HF diet, as compared with controls. Two DGGE bands with reduced intensities in HF-fed mice were identified as representing Lactobacillus gasseri and an uncultured Bacteroides species, whereas a band of increased intensity was identified as representing a Clostridium populeti-related species upon sequencing. Quantitative real-time PCR confirmed a statistically significant 1-log decrease in L. gasseri cell numbers after HF feeding, and revealed a significantly lower level of Bifidobacterium spp. in the control groups after 1 and 2 weeks compared with that in the HF groups. These alterations of intestinal microbiota were not associated with caecum inflammation, as assessed by histological analysis. The observed shifts of specific bacterial populations within the gut may represent an early consequence of increased dietary fat.

The zona-free method of SCNT designed for bovine and pig cloning (Booth et al. 2001; Vajta et al. 2001; Oback et al. 2003) was successfully used for horse (Galli et al. 2003). Although simple and efficient in farm animals, its application in the mouse met several problems (Ribas et al. 2005, 2006). The aim of our work was to produce cloned mice using HM1 embryonic stem (ES)cells adapting a zona-free method. Seven- to 24-week-old superovulated B6D2F1 female mice were used as oocytes donors. Cumulus cells were removed by 0.3% hyaluronidase and the zona pellucida by 0.5% pronase in KSOM-HEPES (KSOM-H) 1 h later (Ribas et al. 2006) or immediately after hyaluronidase treatment at 37°C. The HM1 ES cells were cultured in KnockOut DMEM supplemented with leukemia inhibitory factor and 15% fetal bovine serum with or without 2i (Ying et al. 2008) and were synchronized at M phase by 3 ng mL–1 nocodazole for 3 h before fusion. Only spherical cells were selected for NT. Metaphase II chromosome spindle complexes were removed by micromanipulation in KSOM-H medium with 5 μg mL–1 cytochalasin B. Lectin-treated enucleated oocytes were attached to the donor cells in KSOM-H with nocodazole and fused by 2 pulses of 1.3 kV cm–1 DC for 30 μs in 0.3 M mannitol medium. Following 10- to 15-min incubation in KSOM-H, the fusion was assessed and repeated if the constructs were nonfused. Cloned embryos were activated in 1 mM SrCl2 in Ca2+-free KSOM medium for 2 to 2.5 or 5 to 6 h and cultured in 20-μL KSOM droplets using the well-of-the-well (WOW) method (Vajta et al. 2000) under mineral oil at 37°C and 5% CO2. Day 4 compacted morulae and blastocysts were surgically transferred into the uterus of Day-2.5 pseudopregnant recipients that were sacrificed on Day 19.5 to examine fetal development. The donor mice age was important for oocyte survival: ~16% of oocytes of 7- to 10-week-old mice lysed before or during fusion in 33% of experiments (n experiments = 15), whereas oocytes of older mice were not sensitive to enzymatic treatment and electric impulses even after 3 fusion rounds (n = 19). The time of pronase treatment did not affect oocyte survival, whereas extending the time between hyaluronidase treatment and enucleation revealed self-activation in ~25% of oocytes. The fusion efficiency of ES cells was significantly lower compared with serum-starved fibroblasts (61%, n = 623 v. 100%, n = 80). The duration of SrCl2 treatment did not affect embryo development (cleavage: 82% v. 84%; Day 4 blastocysts: 49% v. 52%). ES cell culture with 2i increased Day 4 blastocyst development (60.7% v. 50.4%; P = 0.07), and their ability to implant (52.6% v. 38.2%; P = 0.06). Moreover, only NT embryos derived from 2i-ES cells developed to term (8.2%, n = 5; P = 0.08), and produced live fetuses (4.9%, n = 3). In light of these results, the fusion of ES cells remains the critical step in the mouse zona-free protocol.Partially supported by grant Superpig from Regione Lombardia.

We investigated the effect of the 52 MAR (matrix attachment region) sequence of chicken lysozyme gene as a possible insulator for the expression of our transgenes in somatic cells to be used for nuclear transfer. With the preliminary purpose to demonstrate a possible positive effect (position or copy number) on the long-term combined expression during in vitro culture, we have created a bicistronic ubiquitous expression vector with (MAR+) or without (MAR–) MAR. The main structure of our constructs is composed of the pCAGGS promoter driving the expression of a reporter gene (enhanced green fluorescent protein, EGFP) followed by a loxable selection cassette (loxP-PGKneo). The MAR region was inserted before the selection cassette. After KpnI digestion, the resulting linearized vectors were purified and subsequently used to transfect adult porcine fibroblast cell lines using the Nucleofector system (Amaxa, Cologne, Germany). Fibroblasts were cultured in DMEM/M199 medium (1:1) + 10% FCS supplemented with 5 ng mL–1 of basic fibroblast growth factor (bFGF). In every experiment, 1 � 106 cells were transfected with 2.5 µg of linearized plasmid and selected for 3 weeks with medium supplemented with 400 µg mL–1 of Geneticin (G418 sulfate, Calbiochem, La Jolla, CA). On Day 8 of G418 selection, we analyzed 150 colonies for each treatment, using fluorescence microscopy with fluorescein isothiocyanate filters. Colonies were classified according to size (large) and cell morphology (small cells without signs of aging). In addition, colonies were classified for uniform GFP expression (uniform), patchy GFP expression (variegated), and no GFP expression (negative). Resistant colonies derived from MAR+ and MAR– vectors, respectively, had 36 (24%), 42 (28%), and 56 (37%) v. 79 (53%), 58 (39%), and 29 (19%) uniform, variegated, and negative GFP. Differences were significant for variegated and negative in MAR+ v. MAR– (chi square, P < 0.05). Thirty-six MAR+ and 42 MAR– colonies uniformly expressing GFP were transferred to 24-well plates and subjected to G418 selection until Day 22, when 7 MAR+ and 15 MAR– clones were still growing in culture. Four of seven MAR+ (57%) and 7/15 MAR– (47%) uniformly expressed high levels of GFP. In conclusion, we found that significantly fewer colonies expressed GFP with the MAR+ vector; however, within the GFP-expressing clones, expression was more uniform. Therefore, we did not find a beneficial effect of MAR sequences on expression in somatic cells during in vitro culture; however, further work is needed to investigate their effect after nuclear transfer and/or on the next generation of cloned transgenic animals.