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Communications Biology May 2022The Tg(Pax6-cre,GFP) (α-Cre) mouse is a commonly used Cre line thought to be retinal-specific. Using targeted locus amplification (TLA), we mapped the insertion site of...
The Tg(Pax6-cre,GFP) (α-Cre) mouse is a commonly used Cre line thought to be retinal-specific. Using targeted locus amplification (TLA), we mapped the insertion site of the transgene, and defined primers useful to deduce zygosity. Further analyses revealed four tandem copies of the transgene. The insertion site mapped to clusters of vomeronasal and olfactory receptor genes. Using R26R and Ai14 Cre reporter mice, we confirmed retinal Cre activity, but also detected expression in Gα olfactory neurons. Most α-Cre olfactory neurons do not express Pax6, implicating the influence of neighboring regulatory elements. RT-PCR and buried food pellet test did not detect any effects of the transgene on flanking genes in the nasal mucosa and retina. Together, these data precisely map α-Cre, show that it does not affect surrounding loci, but reveal previously unanticipated transgene expression in olfactory neurons. The α-Cre mouse can be a valuable tool in both retinal and olfactory research.
Topics: Animals; Integrases; Mice; Mice, Transgenic; Neurons; Retina; Transgenes
PubMed: 35505181
DOI: 10.1038/s42003-022-03379-9 -
Neuropharmacology Jun 2013Diseases of the central nervous system (CNS) have provided enormous opportunities for the therapeutic application of viral vector gene transfer. Adeno-associated virus... (Review)
Review
Diseases of the central nervous system (CNS) have provided enormous opportunities for the therapeutic application of viral vector gene transfer. Adeno-associated virus (AAV) has been the vector of choice in recent clinical trials of neurological disease, including Parkinson's and Alzheimer's disease, due to the safety, efficacy, and stability of AAV gene transfer to the CNS. This review highlights the strategies employed for improving direct and peripheral targeting of therapeutic vectors to CNS tissue, and considers the significance of cellular and tissue transduction specificity, transgene regulation, and other variables that influence achievement of successful therapeutic goals. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'.
Topics: Animals; Dependovirus; Gene Expression; Genetic Therapy; Genetic Vectors; Humans; Injections, Intraventricular; Nervous System Diseases; Transgenes
PubMed: 22465202
DOI: 10.1016/j.neuropharm.2012.03.004 -
Methods in Molecular Biology (Clifton,... 2011Transgenics are powerful mouse models to understand the biological functions of genes. This chapter gives a short overview of the requirements and considerations in...
Transgenics are powerful mouse models to understand the biological functions of genes. This chapter gives a short overview of the requirements and considerations in designing a transgene. In addition, potential important choices that have to be made in advance for the successful designing and generating a transgenic mouse model are discussed. Methods for DNA purification for microinjection are also provided in this chapter.
Topics: Animals; Mice; Mice, Transgenic; Microinjections; Models, Genetic; Promoter Regions, Genetic; Transgenes
PubMed: 21080276
DOI: 10.1007/978-1-60761-974-1_6 -
Plant Physiology Mar 2022Transgene residuals in edited plants affect genetic analysis, pose off-target risks, and cause regulatory concerns. Several strategies have been developed to efficiently...
Transgene residuals in edited plants affect genetic analysis, pose off-target risks, and cause regulatory concerns. Several strategies have been developed to efficiently edit target genes without leaving any transgenes in plants. Some approaches directly address this issue by editing plant genomes with DNA-free reagents. On the other hand, DNA-based techniques require another step for ensuring plants are transgene-free. Fluorescent markers, pigments, and chemical treatments have all been employed as tools to distinguish transgenic plants from transgene-free plants quickly and easily. Moreover, suicide genes have been used to trigger self-elimination of transgenic plants, greatly improving the efficiency of isolating the desired transgene-free plants. Transgenes can also be excised from plant genomes using site-specific recombination, transposition or gene editing nucleases, providing a strategy for editing asexually produced plants. Finally, haploid induction coupled with gene editing may make it feasible to edit plants that are recalcitrant to transformation. Here, we evaluate the strengths and weaknesses of recently developed approaches for obtaining edited plants without transgene residuals.
Topics: Endonucleases; Gene Editing; Genome, Plant; Plants, Genetically Modified; Transgenes
PubMed: 34893903
DOI: 10.1093/plphys/kiab574 -
Journal of Cellular Biochemistry May 2016Activation of Notch1 in osteocytes of Rosa(Notch) mice, where a loxP-flanked STOP cassette and the Nicd coding sequence were targeted to the reverse orientation splice...
Activation of Notch1 in osteocytes of Rosa(Notch) mice, where a loxP-flanked STOP cassette and the Nicd coding sequence were targeted to the reverse orientation splice acceptor (Rosa)26 locus, causes osteopetrosis associated with suppressed Sost expression and enhanced Wnt signaling. To determine whether Sost downregulation mediates the effects of Notch activation in osteocytes, Rosa(Notch) mice were crossed with transgenics expressing Cre recombinase or SOST under the control of the dentin matrix protein (Dmp)1 promoter. Dmp1-SOST transgenics displayed vertebral osteopenia and a modest femoral cancellous and cortical bone phenotype, whereas hemizygous Dmp1-Cre transgenics heterozygous for the Rosa(Notch) allele (Dmp1-Cre;Rosa(Notch)) exhibited osteopetrosis. The phenotype of Notch activation in osteocytes was prevented in Dmp1-Cre;Rosa(Notch) mice hemizygous for the Dmp1-SOST transgene. The effect was associated with downregulated Notch signaling and suppressed Dmp1 and Rosa26 expression. To test whether SOST regulates Notch expression in osteocytes, cortical bone cultures from Dmp1-Cre;Rosa(Notch) mice or from Rosa(Notch) control littermates were exposed to recombinant human SOST. The addition of SOST had only modest effects on Notch target gene mRNA levels and suppressed Dmp1, but not Cre or Rosa26, expression. These findings suggest that prevention of the Dmp1-Cre;Rosa(Notch) skeletal phenotype by Dmp1-SOST is not secondary to SOST expression but to interactions among the Dmp1-SOST and Dmp1-Cre transgenes and the Rosa26 locus. In conclusion, the Dmp1-SOST transgene suppresses the expression of the Dmp1-Cre transgene and of Rosa26.
Topics: Adaptor Proteins, Signal Transducing; Alleles; Animals; Bone Diseases, Metabolic; Extracellular Matrix Proteins; Femur; Gene Expression; Glycoproteins; Integrases; Intercellular Signaling Peptides and Proteins; Mice, Inbred C57BL; Mice, Transgenic; Osteocytes; Osteopetrosis; Protein Binding; RNA, Untranslated; Receptor, Notch1; Reverse Transcriptase Polymerase Chain Reaction; Transgenes; X-Ray Microtomography
PubMed: 26456319
DOI: 10.1002/jcb.25405 -
Transgenic Research Feb 2021The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher...
Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T generation.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T plants, we could remove foreign DNA easily by genetic segregation in the T generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.
Topics: Antigens, Plant; Biolistics; CRISPR-Cas Systems; Crops, Agricultural; Gene Editing; Genome, Plant; Humans; Mutagenesis, Site-Directed; Mutation; Plant Breeding; Plants, Genetically Modified; Soybean Proteins; Glycine max; Transgenes
PubMed: 33386504
DOI: 10.1007/s11248-020-00229-4 -
Xenotransplantation May 2021Thrombosis is a known consequence of intraportal islet transplantation, particularly for xenogeneic islets. To define the origins of thrombosis after islet...
BACKGROUND
Thrombosis is a known consequence of intraportal islet transplantation, particularly for xenogeneic islets. To define the origins of thrombosis after islet xenotransplantation and relate it to early inflammation, we examined porcine islets transplanted into non-human primates using a dual-transplant model to directly compare islet characteristics.
METHODS
α1,3-Galactosyltransferase gene-knockout (GTKO) islets with and without expression of the human complement regulatory transgene CD46 (hCD46) were studied. Biologically inert polyethylene microspheres were used to examine the generic pro-thrombotic effects of particle embolization. Immunohistochemistry was performed 1 and 24 hours after transplantation.
RESULTS
Xeno-islet transplantation activated both extrinsic and intrinsic coagulation pathways. The intrinsic pathway was also initiated by microsphere embolization, while extrinsic pathway tissue factor (TF) and platelet aggregation were more specific to engrafted islets. hCD46 expression significantly reduced TF, platelet, fibrin, and factor XIIIa accumulation in and around islets but did not alter intrinsic factor activation. Layers of TF cells emerged around islets within 24 hours, particularly co-localized with vimentin, and identified as CD3+ and CD68+ cells inflammatory cells.
CONCLUSIONS
These findings detail the origins of thrombosis following islet xenotransplantation, relate it to early immune activation, and suggest a role for transgenic hCD46 expression in its mitigation. Layers of TF-positive inflammatory cells and fibroblasts around islets at 24 hours may have important roles in the progressive events of thrombosis, inflammatory cell recruitment, rejection, and the ultimate outcome of transplanted grafts. These suggest that the strategies targeting these elements could yield more progress toward successful xenogeneic islet engraftment and survival.
Topics: Animals; Heterografts; Inflammation; Islets of Langerhans Transplantation; Swine; Transgenes; Transplantation, Heterologous
PubMed: 33619844
DOI: 10.1111/xen.12680 -
Bone Nov 2016Advancing our understanding of osteoblast biology and differentiation is critical to elucidate the pathological mechanisms responsible for skeletal diseases such as... (Review)
Review
Advancing our understanding of osteoblast biology and differentiation is critical to elucidate the pathological mechanisms responsible for skeletal diseases such as osteoporosis. Histology and histomorphometry, the classical methods to study osteoblast biology, identify osteoblasts based on their location and morphology and ability to mineralize matrix, but do not clearly define their stage of differentiation. Introduction of visual transgenes into the cells of osteoblast lineage has revolutionized the field and resulted in a paradigm shift that allowed for specific identification and isolation of subpopulations within the osteoblast lineage. Knowledge acquired from the studies based on GFP transgenes has allowed for more precise interpretation of studies analyzing targeted overexpression or deletion of genes in the osteoblast lineage. Here, we provide a condensed overview of the currently available promoter-fluorescent reporter transgenic mice that have been generated and evaluated to varying extents. We cover different stages of the lineage as transgenes have been utilized to identify osteoprogenitors, pre-osteoblasts, osteoblasts, or osteocytes. We show that each of these promoters present with advantages and disadvantages. The studies based on the use of these reporter mice have improved our understanding of bone biology. They constitute attractive models to target osteoblasts and help to understand their cell biology.
Topics: Animals; Cell Differentiation; Cell Lineage; Green Fluorescent Proteins; Humans; Luminescent Proteins; Osteoblasts; Osteocytes; Transgenes
PubMed: 27616604
DOI: 10.1016/j.bone.2016.09.004 -
Human Gene Therapy Apr 2014Adenoviruses are efficient gene delivery vectors based on their ability to transduce a wide variety of cell types and drive high-level transient transgene expression.... (Review)
Review
Adenoviruses are efficient gene delivery vectors based on their ability to transduce a wide variety of cell types and drive high-level transient transgene expression. While there have been advances in modifying human adenoviral (HAdV) vectors to increase their safety profile, there are still pitfalls that need to be further addressed. Preexisting humoral and cellular immunity against common HAdV serotypes limits the efficacy of gene transfer and duration of transgene expression. As an alternative, nonhuman AdV (NHAdV) vectors can circumvent neutralizing antibodies against HAdVs in immunized mice and monkeys and in human sera, suggesting that NHAdV vectors could circumvent preexisting humoral immunity against HAdVs in a clinical setting. Consequently, there has been an increased interest in developing NHAdV vectors for gene delivery in humans. In this review, we outline the recent advances and limitations of HAdV vectors for gene therapy and describe examples of NHAdV vectors focusing on their immunogenicity, tropism, and potential as effective gene therapy vehicles.
Topics: Adaptive Immunity; Adenoviridae; Adenoviridae Infections; Animals; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Immunity; Immunity, Innate; Transgenes
PubMed: 24499174
DOI: 10.1089/hum.2013.228 -
Bioinformatics (Oxford, England) Apr 2022Transgene-design is a web application to help design transgenes for use in mammalian studies. It is predicated on the recent discovery that human intronless transgenes...
SUMMARY
Transgene-design is a web application to help design transgenes for use in mammalian studies. It is predicated on the recent discovery that human intronless transgenes and native retrogenes can be expressed very effectively if the GC content at exonic synonymous sites is high. In addition, as exonic splice enhancers resident in intron containing genes may have different utility in intronless genes, these can be reduced or increased in density. Input can be a native gene or a commercially 'optimised' gene. The option to leave in the first intron and to protect or avoid other motifs is also permitted.
AVAILABILITY AND IMPLEMENTATION
Transgene-design is based on a ruby for rails platform. The application is available at https://transgene-design.bath.ac.uk. The code is available under GNU General Public License from GitHub (https://github.com/smuehlh/transgenes).
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Animals; Humans; Software; Introns; Exons; Transgenes; Mutation; Mammals
PubMed: 35244144
DOI: 10.1093/bioinformatics/btac139