Mouse Genomic Fragment Research Articles

ETS-dependent enhancers for endothelial-specific expression of serum/glucocorticoid-regulated kinase 1 during mouse embryo development.

Serum/glucocorticoid-regulated kinase 1 (SGK1) is predominantly expressed in endothelial cells of mouse embryos, and Sgk1 null mice show embryonic lethality due to impaired vascular formation. However, how the SGK1 expression is controlled in developing vasculature remains unknown. In this study, we first identified a proximal endothelial enhancer through lacZ reporter mouse analyses. The mouse Sgk1 proximal enhancer was narrowed down to the 5' region of the major transcription initiation site, while a human corresponding region possessed relatively weak activity. We then searched for distal enhancer candidates using in silico analyses of publicly available databases for DNase accessibility, RNA polymerase association and chromatin modification. A region approximately 500kb distant from the human SGK1 gene was conserved in the mouse, and the mouse and human genomic fragments drove transcription restricted to embryonic endothelial cells. Minimal fragments of both proximal and distal enhancers had consensus binding elements for the ETS transcription factors, which were essential for the responsiveness to ERG, FLI1 and ETS1 proteins in luciferase assays and the endothelial lacZ reporter expression in mouse embryos. These results suggest that endothelial SGK1 expression in embryonic vasculature is maintained through at least two ETS-regulated enhancers located in the proximal and distal regions.

A genetic system for biasing the sex ratio in mice.

Biasing the sex ratio of populations of different organisms, including plants, insects, crustacean, and fish, has been demonstrated by genetic and non-genetic approaches. However, biasing the sex ratio of mammalian populations has not been demonstrated genetically. Here, we provide a first proof of concept for such a genetic system in mammals by crossing two genetically engineered mouse lines. The maternal line encodes a functional Cas9 protein on an autosomal chromosome, whereas the paternal line encodes guide RNAs on the Y chromosome targeting vital mouse genes. After fertilization, the presence of both the Y-encoded guide RNAs from the paternal sperm and the Cas9 protein from the maternal egg targets the vital genes in males. We show that these genes are specifically targeted in males and that this breeding consequently self-destructs solely males. Our results pave the way for a genetic system that allows biased sex production of livestock.

Efficient Depletion of Essential Gene Products for Loss-of-Function Studies in Embryonic Stem Cells.

The development of the CRISPR/Cas9 technology has provided powerful methods to target genetic alterations. However, investigating the function of genes essential for cell survival remains problematic, because genetic ablation of these genes results in cell death. As a consequence, cells recombined at the targeted gene and fully depleted of the gene product cannot be obtained. RNA interference is well suited for the study of essential genes, but this approach often results in a partial depletion of the targeted gene product, which can lead to misinterpretations. We previously developed the pHYPER shRNA vector, a high efficiency RNA interference vector, which is based on a 2.5-kb mouse genomic fragment encompassing the H1 gene. We provide here a pHYPER-based protocol optimized to study the function of essential gene products in mouse embryonic stem cells.

Eukaryotic gene invasion by a bacterial mobile insertion sequence element IS2 during cloning into a plasmid vector

Escherichia coli (E. coli) are commonly used as hosts for DNA cloning and sequencing. Upon transformation of E. coli with recombined vector carrying a gene of interest, the bacteria multiply the gene of interest while maintaining the integrity of its content. During the subcloning of a mouse genomic fragment into a plasmid vector, we noticed that the size of the insert increased significantly upon replication in E. coli. The sequence of the insert was determined and found to contain a novel DNA sequence within the mouse genomic insert. A BLAST search of GenBank revealed the novel sequence to be that of the Insertion Sequence 2 (IS2) element from E. coli that was likely inserted during replication in that organism. Importantly, a detailed search of GenBank shows that the IS2 is present within many eukaryotic nucleotide sequences, and in many cases, has been annotated as being part of the protein. The results of this study suggest that one must perform additional careful analysis of the sequence results using BLAST comparisons, and further verification of gene annotation before submission into the GenBank.

PHYPER, a shRNA Vector for High-Efficiency RNA Interference in Embryonic Stem Cells

RNA interference (RNAi) is a powerful method to generate loss-of-function phenotypes. Plasmid vectors with RNA polymerase III promoters have been developed to express short hairpin RNAs (shRNAs) in mammalian cells. In order to optimize the efficiency of these vectors in embryonic stem (ES) cells, we have constructed and tested several plasmids, based on the H1 promoter; that direct the expression of shRNAs. The original pSUPER vector was used as a reference in this study. This vector drives the expression of shRNAs from a basic 0.2-kb H1 promoter; which exhibits a variable expression when integrated into the genome of ES cells. We used a 2.5-kb mouse genomic fragment containing the H1 promoter to construct a new H1 shRNA vector pHYPER. A comparison of this vector with the basic 0.2-kb H1 vector showed that pHYPER directs the synthesis of higher amounts of shRNAs. Using epifluorescence and fluorescent-activated cell sorting (FACS) analysis, we demonstrated that pHYPER is 4-fold more active than the 0.2-kb H1-based vector after integration into the genome of mouse ES cells. We provide a new, improved H1 shRNA vector that is optimized for both transient transfection studies and the generation of stable ES cell lines.

Deletion of Genes From the Mouse Genome Using Cre/loxP Technology.

The steps required to delete genes from the mouse genome are illustrated by showing how a cluster of three flavin-containing monooxygenase (Fmo) genes (Fmo1, Fmo2, and Fmo4) were deleted from mouse chromosome 1. Such large deletions are accomplished using loxP/Cre recombinase technology. Genomic clones corresponding to the genes to be deleted are first isolated, and then appropriate genomic fragments are cloned into vectors containing a loxP site. This produces targeting vectors, which are electroporated into mouse embryonic stem (ES) cells to allow a homologous recombination event to take place between the mouse genomic fragment, present within the vector, and the homologous sequences in the ES cell genome. Screening of ES cells for recombinants in which loxP sites have been inserted on either side of the gene cluster to be deleted is described. Recombination by Cre recombinase to produce ES cell lines carrying the deletion on chromosome 1 is also described.

The mouse sphingomyelin synthase 1 ( SMS1) gene is alternatively spliced to yield multiple transcripts and proteins

Sphingomyelin synthase 1 (SMS1) is a recently identified 413-residue protein that plays a critical role in sphingolipid metabolism by catalyzing the conversion of ceramide and phosphatidylcholine to sphingomyelin and diacylglycerol (DAG). We have previously reported the isolation of a mouse SMS1 encoding cDNA that contains a unique 5′ UTR sequence. Three other mouse SMS1 cDNAs that differed in their 5′ and 3′ non-coding sequences were present in GenBank. In order to ascertain the origin of the unique 5′ and 3′ UTR sequences, we analyzed the structure of the mouse SMS1 gene. Analysis of the four different SMS1 cDNA sequences and of the corresponding mouse genomic fragment revealed that the SMS1 gene consists of 16 exons that are alternatively spliced to produce 4 different mRNAs ( SMS1α1, SMS1α2, SMS1β and SMS1γ) and 3 different proteins (SMS1α, SMS1β and SMS1γ). RT–PCR was used to demonstrate that all four SMS1 cDNAs represent expressed transcripts that show distinctly different tissue distributions. Transcripts for SMS1α1, SMS1α2 and SMS1β were found to increase in response to the pro-apoptotic effects of TNF-α. Finally, using a yeast-based assay, we confirmed that SMS1α prevents the growth inhibitory effects of Bax but SMS1β neither prevents nor enhances the effects of Bax or of SMS1α. Taken together these results demonstrate the complexity of SMS1 gene structure, expression and function.

InteractionIn Vitroof Type III Intermediate Filament Proteins with Higher Order Structures of Single-Stranded DNA, Particularly with G-Quadruplex DNA

Cytoplasmic intermediate filament (cIF) proteins interact strongly with single-stranded (ss) DNAs and RNAs, particularly with G-rich sequences. To test the hypothesis that this interaction depends on special nucleotide sequences and, possibly, higher order structures of ssDNA, a random mixture of mouse genomic ssDNA fragments generated by a novel "whole ssDNA genome PCR" technique via RNA intermediates was subjected to three rounds of affinity binding to in vitro reconstituted vimentin IFs at physiological ionic strength with intermediate PCR amplification of the bound ssDNA segments. Nucleotide sequence and computer folding analysis of the vimentin-selected fragments revealed an enrichment in microsatellites, predominantly of the (GT)n type, telomere DNA, and C/T-rich sequences, most of which, however, were incapable of folding into stable stem-loop structures. Because G-rich sequences were underrepresented in the vimentin-bound fraction, it had to be assumed that such sequences require intramolecular folding or lateral assembly into multistrand structures to be able to stably interact with vimentin, but that this requirement was inadequately fulfilled under the conditions of the selection experiment. For that reason, the few vimentin-selected G-rich ssDNA fragments and a number of telomere models were analyzed for their capacity to form inter- and intramolecular Gquadruplexes (G4 DNAs) under optimized conditions and to interact as such with vimentin and its type III relatives, glial fibrillary acidic protein, and desmin. Band shift assays indeed demonstrated differential binding of the cIF proteins to parallel four-stranded G4 DNAs and, with lower affinity, to bimolecular G'2 and unimolecular G'4 DNA configurations, whereby the transition regions from four- to single-strandedness played an additional role in the binding reaction. In this respect, the binding activity of cIF proteins was comparable with that toward other noncanonical DNA structures, like ds/ss DNA forks, triplex DNA, four-way junction DNA and Z-DNA, which also involve configurational transitions in their interaction with the filament proteins. Association of the cIF proteins with the corresponding nonfolded G-rich ssDNAs was negligible. Considering the almost universal involvement of ssDNA regions and G-quadruplexes in nuclear processes, including DNA transcription and recombination as well as telomere maintenance and dynamics, it is plausible to presume that cIF proteins as complementary constituents of the nuclear matrix participate in the cell- and tissue-specific regulation of these processes.

Gene disruption/knock-in analysis of mONT3: vector construction by employing both in vivo and in vitro recombinations

We report the isolation, spatial/temporal expression and gene disruption phenotype of the mouse ONT3 (mONT3) gene, which encodes a novel secreted signaling protein belonging to the Olfactomedin/Noelin/Tiarin family. During early embryogenesis, mONT3 is detected in the proximal region of the allantois on embryonic day (E) 7.25, in the lateral plate mesoderm on E 8.0 and in the CNS and heart on E 8.5. The homozygous mutant is born normal and fertile. For the expression pattern and loss-of-function analyses, we have successfully generated the LacZ-knock-in targeting vector directly from BACs carrying mouse genomic fragments by combining in vivo and in vitro recombination techniques. This approach enables rapid and reproducible construction of the fully functional vectors within two weeks without the use of restriction enzyme digestion and ligation, or the use of PCR-amplification of large genomic fragments. In addition, this method is applicable to rapid generation of transgenic vectors, demonstrating its versatility in reverse genetic studies.

Enteroendocrine cell expression of a cholecystokinin gene construct in transgenic mice and cultured cells.

CCK is predominantly expressed in subsets of endocrine cells in the intestine and neurons in the brain. We evaluated the expression of a CCK gene construct in transgenic mice and cultured cells to identify a genomic region that directs correct tissue- and cell-specific expression in enteroendocrine cells. The CCKL1 transgene contained 6.4 kb of mouse Cck fused to lacZ. Expression was evaluated in three transgenic lines (J11, J12, J14) by measurement of beta-galactosidase in tissue homogenates and frozen sections. Correct tissue-specific expression was observed, with beta-galactosidase activity detected in intestine and brain. However, there were differences seen in cell-specific expression in the intestine. Line J14 exhibited expression in CCK-endocrine cells, with expressing cells arising at the normal time during fetal development. However, transgene expression in line J12 intestine was limited to neurons of the enteric nervous system, which reflect an early fetal expression pattern for CCK. Analysis of an additional 15 transgenic founder mice demonstrated intestinal expression in 40% of transgenics, with expressing mice following either an endocrine cell pattern or a neuronal pattern in approximately equal numbers. CCKL1 transfection analysis in cultured cells also demonstrated enteroendocrine cell expression, with 100-fold enhanced activity in the enteroendocrine cell line STC-1 compared with nonendocrine cell lines. The results suggest that the minimal cis-regulatory DNA elements necessary for appropriate CCK expression in enteroendocrine cells reside within the 6.4-kb mouse genomic fragment.

Murine cystathionine γ-lyase: complete cDNA and genomic sequences, promoter activity, tissue distribution and developmental expression

Cystathionine gamma-lyase (CSE) is the last key enzyme in the trans-sulphuration pathway for biosynthesis of cysteine from methionine. Cysteine could be provided through diet; however, CSE has been shown to be important for the adequate supply of cysteine to synthesize glutathione, a major intracellular antioxidant. With a view to determining physiological roles of CSE in mice, we report the sequence of a complete mouse CSE cDNA along with its associated genomic structure, generation of specific polyclonal antibodies, and the tissue distribution and developmental expression patterns of CSE in mice. A 1.8 kb full-length cDNA containing an open reading frame of 1197 bp, which encodes a 43.6 kDa protein, was isolated from adult mouse kidney. A 35 kb mouse genomic fragment was obtained by lambda genomic library screening. It contained promoter regions, 12 exons, ranging in size from 53 to 579 bp, spanning over 30 kb, and exon/intron boundaries that were conserved with rat and human CSE. The GC-rich core promoter contained canonical TATA and CAAT motifs, and several transcription factor-binding consensus sequences. The CSE transcript, protein and enzymic activity were detected in liver, kidney, and, at much lower levels, in small intestine and stomach of both rats and mice. In developing mouse liver and kidney, the expression levels of CSE protein and activity gradually increased with age until reaching their peak value at 3 weeks of age, following which the expression levels in liver remained constant, whereas those in kidney decreased significantly. Immunohistochemical analyses revealed predominant CSE expression in hepatocytes and kidney cortical tubuli. These results suggest important physiological roles for CSE in mice.

The genomic organization of mouse resistin reveals major differences from the human resistin: functional implications

The resistin gene is a potential candidate for the etiology of insulin resistance and type 2 diabetes and has been implicated as the molecular link between type 2 diabetes and obesity. Unlike the mouse resistin, expression of the human resistin appears to be regulated differently. We report comparative analyses of the mouse and human genomic fragments encoding the resistin gene. At the amino acid level the two proteins exhibit 59% identity. While at the mRNA level the human resistin shows 64.4% sequence identity with its mouse counterpart, the mouse resistin genomic sequence displays only 46.7% sequence identity with the human resistin and is almost three times bigger than the human resistin. The intronic sequences per se displayed the least identities (28.7%), however the intron boundaries were highly conserved between human and mouse. The mouse resistin carries a very large intron in the 3′ UTR, which has a number of regulatory sequences possibly involved in differential gene expression. Of particular significance is the presence of a PPAR/RXR heterodimer binding site within intron X (IntX-PPRE) which may possibly confer TZD responsiveness. Oligonucleotides carrying the authentic PPAR/RXR binding element (Aco-PPRE) as well as IntX-PPRE specifically bound factors (PPAR/RXR heterodimers) present in differentiated 3T3-L1 adipocyte cells in an electrophoretic mobility shift assay. IntX-PPRE oligonucleotide modulated the expression of the luciferase reporter gene in transient transfection assays using 3T3-L1 cells.

Cloning and Characterization of the Murine PKC α Promoter: Identification of a Retinoic Acid Response Element

Protein kinase C (PKC) is a family which consists of multiple isoforms whose distinct physiological roles within the cell are unknown. We have previously demonstrated that levels of PKC α mRNA, protein, and enzyme activity in B16 melanoma cells can be modulated by retinoic acid. We investigated this regulation by cloning and characterizing the promoter region of the murine PKC α gene. A 13 kb mouse genomic fragment containing the 5′ flanking region, first exon, and first intron was isolated and sequenced. Two transcription initiation sites were identified at 919 and 925 bp upstream from the translation start site. The promoter region contained a TATA-like box at −93 bp upstream of the transcription start site, but no CAAT box. Promoter activity differed between cell lines and correlated with the levels of PKC α expressed in these cell lines. Reporter gene assays showed that the region between −179 and −452 bp likely contains a silencer element(s). The promoter activity of a −179 bp fragment in B16 cells was stimulated twofold by retinoic acid. Within this region (−93 to −65 bp) there is a retinoic acid response element. An oligonucleotide spanning this region specifically bound exogenous RAR-RXR heterodimers and endogenous RAR from B16 nuclear extracts. These results suggest that retinoic acid increases PKC α gene expression in B16 cells, at least in part, through direct transcriptional stimulation of its promoter.

Cloning and characterization of 5′-flanking region of mouse non-selective cation channel 1

We have previously cloned mouse non-selective cation channel 1 (mNSC1) cDNA inducing cation current, from a mouse insulin secreting β-cell line, MIN6. The current has characteristics of the Ca 2+-activated non-selective (CAN) cation channel, and the mRNA is localized in the brain, heart, and lung. To understand the molecular mechanisms of the transcriptional regulation, we have cloned and characterized the 5′-flanking region of mNSC1. By the PCR method, we obtained 987 bp of mouse genomic fragment. The computer program-based analysis revealed that it contained several consensus motifs; insulin responsive element (IRE), AP-2, PEA3, and GC box-like region. But there were neither typical TATA box nor CAAT box. Primer extension analysis and RNase protection assay were performed to identify the transcription start site. Transient transfection analyses using a series of 5′-end deletion and reporter gene constructs with CHO and LA-4 cells demonstrated some relatively active regions. The significantly active border correlated with IRE consensus with CHO cells. This observation may support that CAN current is activated by insulin.

The LIM domains of hic-5 protein recognize specific DNA fragments in a zinc-dependent manner in vitro.

hic-5 protein is a member of the LIM protein family, containing four LIM domains in its C-terminal region. It is mainly localized in focal adhesions and shows striking similarity to paxillin in its LIM domains, although the function of these LIM domains has remained elusive. In the present study, we found that full-length and the C-terminal half of hic-5 protein, including four LIM domains, bound to DNA in a zinc-dependent manner in vitro . Mouse genomic fragments that specifically bound to the hic-5 protein were isolated by successive rounds of hic-5 protein-DNA complex immunoprecipitation and PCR amplification. Seven independent clones were isolated, which contained high amounts of G+A and/or a long A/T tract. A DNA binding protein blot assay revealed the specificity of the interaction between hic-5 protein and the DNA fragment. Using a series of truncated forms of the hic-5 LIM domains, each of the four LIM domains was found to contribute to DNA binding in a distinctive manner.

Comparative mapping on the mouse X Chromosome defines a myotubular myopathy equivalent region

The gene for X-linked myotubular myopathy (MTM1) has been localized to a 300-kb critical region in human Xq28 between IDS and GABRA3. As part of an effort to clone this gene, we developed a YAC contig on the mouse X Chromosome (Chr) which includes loci homologous to those within the human MTM1 critical region. The murine contig consists of 18 YACs and spans 2.5-3.0 Mb. We have aligned the human and murine physical maps by isolating conserved mouse genomic fragments, including CpG islands and trapped exons. We believe that the simultaneous isolation of genes from both mouse and human and continued comparative mapping will prove helpful in the eventual identification of MTM1 and other genes in the region.

Assignment of the human prosaposin gene (PSAP) to 10q22.1 by fluorescence in situ hybridization. Giraffidae, okapi (Okapiajohnstoni), and giraffe (Giraffa camelopardalis): evidence for ancestral telomeres at the okapi polymorphic rob (4;26) fusion site.

The human prosaposin gene (PSAP) was previously localized to 10q21-->q22 by isotopic in situ hybridization using a human prosaposin cDNA as a probe. The present study, using fluorescence in situ hybridization with a mouse genomic prosaposin fragment as probe, confirms the localization of PSAP and precisely maps it to band 10q22.1.

The Mouse APLP2 Gene: CHROMOSOMAL LOCALIZATION AND PROMOTER CHARACTERIZATION

Senile plaques are primarily comprised of deposits of the beta-amyloid precursor-like proteins APLP1 and APLP2. proteins (APPs). APP is a member of a gene family, including amyloid precursor-like proteins APLP1 and APLP2. Using interspecific mouse backcross mapping, we localized the mouse APLP2 gene to the promixmal region of mouse chromosome 9, syntenic with a region of human 11q. We cloned an approximately 1.2-kilobase mouse genomic fragment containing the APLP2 gene promoter. The APLP2 promoter lacks a typical TATA box, is GC-rich, and contains several sequences for transcription factor binding. S1 nuclease protection analysis revealed the presence of multiple transcription start sites. The lack of a TATA box, the presence of a high GC content, and multiple transcription start sites place the APLP2 promoter in the class of promoters of "housekeeping genes." Regulatory regions within the promoter were assayed by transfection of mouse N2a and Ltk- cells with constructs containing progressive 5'-deletions of the APLP2 promoter fused to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. A minimal region that includes sequences 99 bp upstream of the predominant transcription start site of the APLP2 promoter was sufficient to direct high levels of CAT expression.

In male mouse germ cells, copper-zinc superoxide dismutase utilizes alternative promoters that produce multiple transcripts with different translation potential.

Copper-zinc superoxide dismutase (SOD-1) is an enzyme that is widely expressed in eukaryotic cells and performs a vital role in protecting cells against free radical damage. In mouse testis, three different sizes of SOD-1 mRNAs of about 0.73, 0.80, and 0.93 kilobases (kb) are detected. The 0.73-kb mRNA is found in early stages of male germ cells and in all somatic tissues. The mRNAs of 0.80 and 0.93 kb are exclusively detected in post-meiotic germ cells. RNase H digestions and Northern blot analyses reveal that the three SOD-1 mRNAs are derived from two transcripts, a ubiquitously expressed transcript and a post-meiotic transcript, which differ by 114-120 nucleotides. RNase protection assays demonstrate that the additional nucleotides present in the post-meiotic mRNA are solely in the 5'-untranslated region. Using a probe derived from the 5'-untranslated region of the 0.93-kb SOD-1 mRNA, we have established that it originates from an alternative upstream promoter contiguous with the somatic SOD-1 promoter. Polysomal gradient analysis of the three mouse testis SOD-1 mRNAs reveals that the 0.93-kb SOD-1 mRNA is primarily non-polysomal, while the 0.80- and 0.73-kb SOD-1 mRNAs are mostly polysome associated. A faster migrating form of the 0.93-kb SOD-1 mRNA is present on polysomes as a result of partial deadenylation. In a cell-free translation system, the 0.73-kb SOD-1 mRNA translates about 2-fold more efficiently than the 0.93-kb SOD-1 mRNA. These data demonstrate that male germ cells transcribe two size classes of SOD-1 mRNAs with different translation potential by utilizing two different promoters, post-meiotic SOD-1 mRNAs undergo adenylation changes, and one of the post-meiotic SOD-1 mRNAs is transcribed during mid-spermiogenesis and translated days later in a partially deadenylated form.

Two murine GSTpi genes are arranged in tandem and are differentially expressed.

An 8-kilobase mouse genomic fragment containing two intact glutathione S-transferase (GST) genes has been isolated from a mouse lambda genomic library. Each of the genes (designated mGSTpiA and mGSTpiB) is less than 3 kilobases in size and is comprised of seven exons that give rise to a 630-base pair open reading frame encoding 209 amino acids. The deduced amino acid sequences of the gene products differ in only 6 amino acids at positions 10, 11, 89, 104, 106, and 109. These two genes are highly homologous to rat GST-P and to a lesser extent to human GST-pi. Northern blot analysis of mRNAs from a variety of mouse tissues demonstrated that mGSTpiB is ubiquitously expressed, whereas mGST-piA is more selectively expressed in gallbladder, colon, heart, and skeletal muscle. Primer extension analysis revealed four potential transcription start sites in mGST-piB and one in mGSTpiA. Although both genes were expressed in vitro and in vivo only mGSTpiB product metabolized 1-chloro-2,4-dinitrobenzene, a common GST substrate. Further in vitro expression studies of three chimeric mGSTpi genes suggested that one or both of the amino acids at positions 10 and 11 of mouse GSTpi enzymes are important for the enzyme's ability to metabolize 1-chloro-2,4-dinitrobenzene.