scholarly journals Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene.

1996 ◽  
Vol 93 (9) ◽  
pp. 3932-3936 ◽  
Author(s):  
Y. Wang ◽  
L. A. Krushel ◽  
G. M. Edelman
Keyword(s):  
Dna Recombination ◽  
Cre Recombinase ◽  
Recombinase Gene

scholarly journals A novel mouse line with epididymal initial segment-specific expression of Cre recombinase driven by the endogenous Lcn9 promoter

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254802
Author(s):  
Qian-qian Gong ◽  
Xiao Wang ◽  
Zhi-lin Dou ◽  
Ke-yi Zhang ◽  
Xiang-guo Liu ◽  
...  
Keyword(s):  
Initial Segment ◽  
Stop Codon ◽  
Dna Recombination ◽  
Cre Recombinase ◽  
Functional Study ◽  
The Novel ◽  
Mouse Line ◽  
Specific Expression ◽  
Reporter Mice

Spermatozoa released from testes undergo a maturation process and acquire the capacity to fertilize ova through epididymal transit. The epididymis is divided into four regions, each with unique morphology, gene profile, luminal microenvironment and distinct function. To study the functions of relevant genes in the epididymal initial segment (IS), a novel IS-specific mouse model, Lcn9-Cre knock-in (KI) mouse line was generated via CRISPR/Cas9 technology. The TAG stop codon was replaced by a 2A-NLS-Cre cassette, resulting in the co-expression of Lcn9 and Cre recombinase. IS-specific Cre expression was first observed from postnatal day 17. Using the Rosa26tdTomato reporter mice, the Cre-mediated DNA recombination was detected exclusively in principal cells. The epididymal IS-specific Cre activity in vivo was further confirmed using Lcn9-Cre mice crossed with a mouse strain carrying Tsc1 floxed alleles (Tsc1flox/+). Cre expression did not affect either normal development or male fecundity. Different from any epididymis-specific Cre mice reported previously, the novel Lcn9-Cre mouse line can be used to introduce entire IS-specific conditional gene editing for gene functional study.

scholarly journals Characterization of Cre Recombinase Activity for In Vivo Targeting of Adipocyte Precursor Cells

2014 ◽  
Vol 3 (6) ◽  
pp. 1147-1158 ◽  
Author(s):  
Katherine C. Krueger ◽  
Maria José Costa ◽  
Hongqing Du ◽  
Brian J. Feldman
Keyword(s):  
Cre Recombinase ◽  
Precursor Cells ◽  
Adipocyte Precursor

scholarly journals UV-induced Hyperphosphorylation of Replication Protein A Depends on DNA Replication and Expression of ATM Protein

2001 ◽  
Vol 12 (5) ◽  
pp. 1199-1213 ◽  
Author(s):  
Gregory G. Oakley ◽  
Lisa I. Loberg ◽  
Jiaqin Yao ◽  
Mary A. Risinger ◽  
Remy L. Yunker ◽  
...  
Keyword(s):  
Dna Replication ◽  
Uv Radiation ◽  
Excision Repair ◽  
Genetic Disorder ◽  
Protein A ◽  
Dna Recombination ◽  
Delayed Response ◽  
Replication Intermediates

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4–8 h) to UV radiation (10–30 J/m2). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

Production and analysis of mutant mice carrying the Cre recombinase gene inserted into the telencephalin gene

1998 ◽  
Vol 31 ◽  
pp. S312
Author(s):  
Kazuhiro Nakamura ◽  
Hisashi Mori ◽  
Yuji Kiyama ◽  
Takeshi Yagi ◽  
Toshiya Manabe ◽  
...  
Keyword(s):  
Cre Recombinase ◽  
Mutant Mice ◽  
Recombinase Gene

scholarly journals Host Cell DNA Repair Pathways in Adeno-Associated Viral Genome Processing

2006 ◽  
Vol 80 (21) ◽  
pp. 10346-10356 ◽  
Author(s):  
Vivian W. Choi ◽  
Douglas M. McCarty ◽  
R. Jude Samulski
Keyword(s):  
Dna Repair ◽  
Dna Recombination ◽  
Recq Helicase ◽  
Cellular Mechanisms ◽  
Adeno Associated Virus ◽  
Mrn Complex ◽  
Dependent Vector ◽  
Cellular Dna ◽  
Dna Maintenance

ABSTRACT Recentstudies have shown that wild-type and recombinant adeno-associated virus (AAV and rAAV) genomes persist in human tissue predominantly as double-stranded (ds) circular episomes derived from input linear single-stranded virion DNA. Using self-complementary recombinant AAV (scAAV) vectors, we generated intermediates that directly transition to ds circular episomes. The scAAV genome ends are palindromic hairpin-structured terminal repeats, resembling a double-stranded break repair intermediate. Utilizing this substrate, we found cellular DNA recombination and repair factors to be essential for generating circular episomal products. To identify the specific cellular proteins involved, the scAAV circularization-dependent vector was used as a reporter in 19 mammalian DNA repair-deficient cell lines. The results show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient scAAV genome circularization. We further demonstrated that the scAAV genome requires ATM and DNA-PKCS, but not NBS1, to efficiently convert to a circular form in nondividing cells in vivo using transgenic mice. These studies identify specific pathways involved for further elucidating viral and cellular mechanisms of DNA maintenance important to the viral life cycle and vector utilizations.

scholarly journals Mouse models for BRAF-induced cancers

2007 ◽  
Vol 35 (5) ◽  
pp. 1329-1333 ◽  
Author(s):  
C. Pritchard ◽  
L. Carragher ◽  
V. Aldridge ◽  
S. Giblett ◽  
H. Jin ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mouse Models ◽  
Human Cancer ◽  
Cre Recombinase ◽  
Genetically Engineered ◽  
Mitogen Activated Protein Kinase ◽  
Braf Mutations ◽  
Single Nucleotide Change

Oncogenic mutations in the BRAF gene are detected in ∼7% of human cancer samples with a particularly high frequency of mutation in malignant melanomas. Over 40 different missense BRAF mutations have been found, but the vast majority (>90%) represent a single nucleotide change resulting in a valine→glutamate mutation at residue 600 (V600EBRAF). In cells cultured in vitro, V600EBRAF is able to stimulate endogenous MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] and ERK phosphorylation leading to an increase in cell proliferation, cell survival, transformation, tumorigenicity, invasion and vascular development. Many of these hallmarks of cancer can be reversed by treatment of cells with siRNA (small interfering RNA) to BRAF or by inhibiting MEK, indicating that BRAF and MEK are attractive therapeutic targets in cancer samples with BRAF mutations. In order to fully understand the role of oncogenic BRAF in cancer development in vivo as well as to test the in vivo efficacy of anti-BRAF or anti-MEK therapies, GEMMs (genetically engineered mouse models) have been generated in which expression of oncogenic BRaf is conditionally dependent on the Cre recombinase. The delivery/activation of the Cre recombinase can be regulated in both a temporal and spatial manner and therefore these mouse models can be used to recapitulate the somatic mutation of BRAF that occurs in different tissues in the development of human cancer. The data so far obtained following Cre-mediated activation in haemopoietic tissue and the lung indicate that V600EBRAF mutation can drive tumour initiation and that its primary effect is to induce high levels of cyclin D1-mediated cell proliferation. However, hallmarks of OIS (oncogene-induced senescence) are evident that restrain further development of the tumour.

Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

2001 ◽  
Vol 114 (4) ◽  
pp. 671-676 ◽  
Author(s):  
E. Gustafsson ◽  
C. Brakebusch ◽  
K. Hietanen ◽  
R. Fassler
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Cre Recombinase ◽  
Embryoid Bodies ◽  
Lymphoid Cells ◽  
Adult Brain ◽  
Specific Gene

Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth, LacZ activity was detected in most endothelial cells within the embryo and of extra-embryonic tissues such as yolk sac and chorioallantoic placenta. Ectopic expression of Cre was observed in approximately 12–20% of the adult erythroid, myeloid and lymphoid cells and in subregions of the adult brain. These results show that the tie-1-Cre transgenic strain can efficiently direct deletion of floxed genes in endothelial cells in vivo.

scholarly journals A Novel Cre Recombinase-MediatedIn VivoMinicircle DNA (CRIM) Vaccine Provides Partial Protection against Newcastle Disease Virus

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
Yanlong Jiang ◽  
Xing Gao ◽  
Ke Xu ◽  
Jianzhong Wang ◽  
Haibin Huang ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Vaccine Development ◽  
Cre Recombinase ◽  
Attractive Alternative ◽  
Vaccine Platform ◽  
Veterinary Vaccine ◽  
Minicircle Dna

ABSTRACTMinicircle DNA (mcDNA), which contains only the necessary components for eukaryotic expression and is thus smaller than traditional plasmids, has been designed for application in genetic manipulation. In this study, we constructed a novel plasmid containing both the Cre recombinase under the phosphoglycerate kinase (PGK) promoter and recombinantlox66andlox71sites located outside the cytomegalovirus (CMV) expression cassette. The strictly controlled synthesis of Cre recombinasein vivomaintained the complete form of the plasmidin vitro, whereas thein vivoproduction of Cre transformed the parental plasmid to mcDNA after transfection. The newly designedCrerecombinase-mediatedin vivomcDNA platform, named CRIM, significantly increased the nuclear entry of mcDNA, followed by increased production of mRNA and protein, using enhanced green fluorescent protein (EGFP) as a model. Similar results were also observed in chickens when the vaccine was delivered by the regulated-delayed-lysisSalmonellastrain χ11218, where significantly increased production of EGFP was observed in chicken livers. Then, we used the HN gene of genotype VII Newcastle disease virus as an antigen model to construct the traditional plasmid pYL43 and the novel mcDNA plasmid pYL47. After immunization, our CRIM vaccine provided significantly increased protection against challenge compared with that of the traditional plasmid, providing us with a novel mcDNA vaccine platform.IMPORTANCEMinicircle DNA (mcDNA) has been considered an attractive alternative to DNA vaccines; however, the relatively high cost and complicated process of purifying mcDNA dramatically restricts the application of mcDNA in the veterinary field. We designed a novelin vivomcDNA platform in which the complete plasmid could spontaneously transform into mcDNAin vivo. In combination with the regulated-delayed-lysisSalmonellastrain, the newly designed mcDNA vaccine provides us with an elegant platform for veterinary vaccine development.

scholarly journals Variable Lipoprotein Genes of Mycoplasma agalactiae Are Activated In Vivo by Promoter Addition via Site-Specific DNA Inversions

2003 ◽  
Vol 71 (7) ◽  
pp. 3821-3830 ◽  
Author(s):  
Ravenna Flitman-Tene ◽  
Sigalit Mudahi-Orenstein ◽  
Sharon Levisohn ◽  
David Yogev
Keyword(s):  
Dna Recombination ◽  
Small Ruminants ◽  
Upstream Region ◽  
Pcr Analysis ◽  
Phase Variable ◽  
Multiple Sequence ◽  
Site Specific ◽  
Mycoplasma Agalactiae ◽  
Variable Expression

ABSTRACT Mycoplasma agalactiae, the etiological agent of contagious agalactia of small ruminants, has a family of related genes (avg genes) which encode surface lipoprotein antigens that undergo phase variation. A series of 13 M. agalactiae clonal isolates, obtained from one chronically infected animal over a period of 7 months, were found to undergo major rearrangement events within the avg genomic locus. We show that these rearrangements regulate the phase-variable expression of individual avg genes. Northern blot analysis and reverse transcription-PCR showed that only one avg gene is transcribed, while the other avg genes are transcriptionally silent. Sequence analysis and primer extension experiments with two M. agalactiae clonal isolates showed that a specific 182-bp avg 5′ upstream region (avg-B2) that is present as a single chromosomal copy serves as an active promoter and exhibits a high level of homology with the vsp promoter of the bovine pathogen Mycoplasma bovis. PCR analysis showed that each avg gene is associated with the avg-B2 promoter in a subpopulation of cells that is present in each subclone. Multiple sequence-specific sites for DNA recombination (vis-like), which are presumably recognized by site-specific recombinase, were identified within the conserved avg 5′ upstream regions of all avg genes and were found to be identical to the recombination sites of the M. bovis vsp locus. In addition, a gene encoding a member of the integrase family of tyrosine site-specific recombinases was identified adjacent to the variable avg locus. The molecular genetic basis for avg phase-variable expression appears to be mediated by site-specific DNA inversions occurring in vivo that allow activation of a silent avg gene by promoter addition. A model for the control of avg genes is proposed.

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