E. Lader - Academia.edu (original) (raw)

Papers by E. Lader

Antiviral Therapy

Objectives To identify and characterize the siRNA duplexes that are effective for inhibition of S... more Objectives To identify and characterize the siRNA duplexes that are effective for inhibition of SARS-CoV infection and replication in the non-human primate cells. This in vitro study will serve as the foundation for development of novel anti-SARS therapeutics. Methods 48 siRNA sequences were designed for targeting regions throughout entire SARS-CoV genome RNA including open-reading frames for several key proteins. Chemically synthesized siRNA duplexes were transfected into foetal rhesus kidney (FRhK-4) cells prior to or after SARS-CoV infection. The inhibitory effects of the siRNAs were evaluated for reductions of intracellular viral genome copy number and viral titres in the cell culture medium measured by Q-RT-PCR and CPE-based titration, respectively. Four siRNA duplexes were found to achieve potent inhibition of SARS-CoV infection and replication. A prolonged prophylactic effect of siRNA duplexes with up to 90% inhibition that lasted for at least 72 h was observed. Combination o...

Developmental Biology, 1995

A new phenotype mapping to the t-complex, which is designated Brachyury the Second (T2), is chara... more A new phenotype mapping to the t-complex, which is designated Brachyury the Second (T2), is characterized by a slightly shortened tail in heterozygotes and homozygous failure to form an organized notochord with subsequent abnormal development of posterior somites and neural tube. The phenotype of T2 superficially resembles that of Brachyury; however, there are several important differences. Brachyury homozygotes fail to make posterior somites, notochord, floor plate, and a placental connection, resulting in death by 10.5 days of development. In contrast, T2 homozygotes make posterior somites, scattered notochord cells, and floorplate and achieve an allantoic connection. However, despite making a maternal connection, T2 homozygotes cease development at E11.5 and die soon after. We have cloned and analyzed the transgene insertion site, which maps within 100 kb of the Brachyury gene, but does not seem to physically interrupt nor affect transcription from that locus. The existence of a second gene mapping near Brachyury and affecting the same developmental processes was alluded to over 50 years ago and has been debated ever since. An embryological description of T2 is presented, as is a discussion of the implications of a single, larger Brachyury locus versus two closely linked genes coordinately regulating axial development.

Nucleic Acids Research, 2007

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) ... more Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (800 siRNAs, 400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.

The Journal of laboratory and clinical medicine, 1984

Antiserum to cobalamin was raised in rabbits by immunization with the monocarboxyl derivative of ... more Antiserum to cobalamin was raised in rabbits by immunization with the monocarboxyl derivative of cyanocobalamin coupled to albumin. The antiserum was treated to remove transcobalamin II and transcobalamin I. The partially purified antibody bound free cyano[57Co]cobalamin, but not the vitamin precoupled to the transcobalamins. Cyano[57Co]cobalamin bound by the antiserum eluted from Sephadex G-200 as a single peak with a mol wt of 160,000 and was precipitated by goat anti-rabbit gamma globulin, indicating that the vitamin was bound to an IgG immunoglobulin. Unlabeled cyanocobalamin and hydroxocobalamin competitively inhibited the binding of cyano[57Co]cobalamin to this antibody. Neither adenosylcobalamin, in a similar concentration range, nor cyanocobinamide at a concentration 30-fold greater than the tracer cobalamin competed appreciably with the binding of cyano[57Co] cobalamin. The association constant for the interaction of the antibody with cyanocobalamin and cyanocobinamide was ...

Genetics, 1998

The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation i... more The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation in T2 disrupts notochord development, pointing to the existence of a second T/t complex gene involved in axis development. T2 encodes a novel protein that is disrupted by an insertion in T2(Bob) mice. Sequence analysis of T2 from several t haplotypes shows that they all share the same changed stop codon, and, thus, T2 is a candidate gene for the t complex tail interaction factor. T1, T2, and the unlinked t-int are distinct and unrelated loci, and mutations in these genes do not complement one another genetically. Either their products interact in the same pathway during the genesis of the embryonic axis, or the T/t region itself is truly complex.

EMBO molecular medicine, Feb 18, 2015

There is an urgent need for new tools to combat the ongoing tuberculosis (TB) pandemic. Gene expr... more There is an urgent need for new tools to combat the ongoing tuberculosis (TB) pandemic. Gene expression profiles based on blood signatures have proved useful in identifying genes that enable classification of TB patients, but have thus far been complex. Using real-time PCR analysis, we evaluated the expression profiles from a large panel of genes in TB patients and healthy individuals in an Indian cohort. Classification models were built and validated for their capacity to discriminate samples from TB patients and controls within this cohort and on external independent gene expression datasets. A combination of only four genes distinguished TB patients from healthy individuals in both cross-validations and on separate validation datasets with very high accuracy. An external validation on two distinct cohorts using a real-time PCR setting confirmed the predictive power of this 4-gene tool reaching sensitivity scores of 88% with a specificity of around 75%. Moreover, this gene signatu...

Scientific reports, 2015

Our objective was to identify microRNA (miRNA) biomarkers of drug-induced liver and kidney injury... more Our objective was to identify microRNA (miRNA) biomarkers of drug-induced liver and kidney injury by profiling the circulating miRNome in patients with acetaminophen overdose. Plasma miRNAs were quantified in age- and sex-matched overdose patients with (N = 27) and without (N = 27) organ injury (APAP-TOX and APAP-no TOX, respectively). Classifier miRNAs were tested in a separate cohort (N = 81). miRNA specificity was determined in non-acetaminophen liver injury and murine models. Sensitivity was tested by stratification of patients at hospital presentation (N = 67). From 1809 miRNAs, 75 were 3-fold or more increased and 46 were 3-fold or more decreased with APAP-TOX. A 16 miRNA classifier model accurately diagnosed APAP-TOX in the test cohort. In humans, the miRNAs with the largest increase (miR-122-5p, miR-885-5p, miR-151a-3p) and the highest rank in the classifier model (miR-382-5p) accurately reported non-acetaminophen liver injury and were unaffected by kidney injury. miR-122-5p...

The Journal of laboratory and clinical medicine, 1984

Antiserum to cobalamin was raised in rabbits by immunization with the monocarboxyl derivative of ... more Antiserum to cobalamin was raised in rabbits by immunization with the monocarboxyl derivative of cyanocobalamin coupled to albumin. The antiserum was treated to remove transcobalamin II and transcobalamin I. The partially purified antibody bound free cyano[57Co]cobalamin, but not the vitamin precoupled to the transcobalamins. Cyano[57Co]cobalamin bound by the antiserum eluted from Sephadex G-200 as a single peak with a mol wt of 160,000 and was precipitated by goat anti-rabbit gamma globulin, indicating that the vitamin was bound to an IgG immunoglobulin. Unlabeled cyanocobalamin and hydroxocobalamin competitively inhibited the binding of cyano[57Co]cobalamin to this antibody. Neither adenosylcobalamin, in a similar concentration range, nor cyanocobinamide at a concentration 30-fold greater than the tracer cobalamin competed appreciably with the binding of cyano[57Co] cobalamin. The association constant for the interaction of the antibody with cyanocobalamin and cyanocobinamide was ...

The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T ). A mutation ... more The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T ). A mutation in T2 disrupts notochord development, pointing to the existence of a second T/t complex gene involved in axis development. T2 encodes a novel protein that is disrupted by an insertion in T2 Bob mice. Sequence analysis of T2 from several t haplotypes

The HZ5-feline sarcoma virus (FeSV) is a new acute transforming feline retrovirus which was isola... more The HZ5-feline sarcoma virus (FeSV) is a new acute transforming feline retrovirus which was isolated from a multicentric fibrosarcoma of a domestic cat. The HZ5-FeSV transforms fibroblasts in vitro and is replication defective. A biologically active integrated HZ5-FeSV provirus was molecularly cloned from cellular DNA of HZ5-FeSV-infected FRE-3A rat cells. The HZ5-FeSV has oncogene homology with the fms sequences of

Molecular and cellular biology, 1991

The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), p... more The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), performs crucial functions at fertilization and in early development. The transcripts encoding mouse ZP2 and ZP3 are coordinately expressed and accumulate in oocytes during a 2-week growth phase prior to ovulation. The 5'-flanking regions of mouse Zp-2 and Zp-3 genes and their human homologs contain five short DNA sequences (4 to 12 bp) that are 60 to 100% identical and are approximately equidistant upstream of the TATAA box in the four genes. Mutation of these five elements (I, IIA, IIB, III, and IV) in Zp-luciferase constructs demonstrates that the 12-bp element IV, positioned approximately 200 bp upstream from the TATAA box, is necessary for high-level expression from the mouse Zp-2 and Zp-3 promoters after microinjection into the nuclei of 50-microns-diameter oocytes. Injection of minimal Zp-3 promoter constructs containing element IV in either orientation also resulted in high le...

Oligonucleotides, 2009

Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that... more Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that generate highly functional siRNAs for gene knockdown. Nevertheless, "difficult-to-silence" target messenger RNAs (mRNAs) still exist for which no functionally validated siRNAs are available. MicroRNA (miRNA) sites in the mRNA 3'UTR, which interact with miRNA-loaded RNA-induced silencing complex (miRISC) for posttranscriptional gene regulation, provide alternative potentially accessible sites for siRNA. To investigate this, we designed siRNAs directed against single putative miRNA sites (misiRNAs) as predicted by miRNA target databases as well as siRNAs against other regions within the 3'UTR of "difficult-to-silence" targets in this proof-of-principle study. Although their design was not fully optimized, the misiRNAs generally caused higher knockdown than previously designed siRNAs for these targets. In general, knockdown by misiRNAs targeting the miRNA seed region was specific for the target mRNA, and misiRNAs targeting 1 nt upstream of miRNA seed region were similarly potent. We also systematically screened the 3'UTR of two mRNA targets using siRNA-tiling experiments. 5' and 3' regions of the p21-activated kinase 6 (PAK6) 3'UTR were found accessible, whereas the middle portion was largely inaccessible for siRNA knockdown. In ribosomal protein S6 kinase (RPS6KB1) 3'UTR, however, only the 5' region was accessible for siRNA knockdown. Detailed analysis of 10 further "difficult-to-silence" targets revealed that siRNA accessibility at the mRNA 3' end is not a general phenomenon, at least in "difficult-to-silence" targets, as we could not detect significant knockdown by siRNAs directed against this region.

…, 2009

Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that... more Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that generate highly functional siRNAs for gene knockdown. Nevertheless,“difficult-to-silence” target messenger RNAs (mRNAs) still exist for which no ...

Developmental Biology, 1993

Cancer Research, 2010

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We have used a nove... more Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We have used a novel real time PCR approach to profile global changes in microRNA expression during adipocytic differentiation of 3T3-L1 fibroblast cells. This profiling strategy makes use of a novel two stage screen resulting in a substantial increase in throughput and decrease in reagent use. In the first step, cDNA is prepared from individual samples of undifferentiated fibroblast, and cells at early and late stages of Adipocyte differentiation. A pooled cDNA sample prepared by pooling equimolar amounts of individual cDNAs was used in an initial profiling experiment to identify microRNAs that are expressed at detectable levels in the experimental system. Profiling was performed in triplicate with real time PCR assays for 591 mouse miRNAs (miRBase v12.0). In the second stage of screening, individual cDNA samples were each analyzed in triplicate using the assays for miRNAs that showed expression in the pooled cDNA sample. Expression data was normalized against a panel of small non-coding RNAs and was expressed as x-fold changes relative to expresion in undifferentiated 3T3L1 cells. Our results indicate that 192 (∼32%) miRNAs described in miRBase v12.0 were detectable in resting and differentiated 3T3-L1 cells, and of these, 38 (or 20%) showed differential regulation during adipogenesis (x-fold change>3.0). When compared to data from previous array-based profiling studies, our screen identified additional differentially regulated miRNAs. In addition to profiling changes in mature miRNA levels, miRNA precursor stem loop transcripts were also quantified to gain insight into post transcriptional regulation (if any) of miRNA biogenesis during adipocyte differentiation. The high specificity and sensitivity of the miScript RT-PCR system, together with a novel sample-pooling strategy makes this system a method of choice for miRNA expression profiling. Pooled primary profiling can substantially reduce the total number of PCR reactions which need to be performed and increases assay throughput. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3033.

Developmental biology, 1993

Molecular and cellular …, 1991

The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), p... more The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), performs crucial functions at fertilization and in early development. The transcripts encoding mouse ZP2 and ZP3 are coordinately expressed and accumulate in oocytes during a 2-week growth phase prior to ovulation. The 5'-flanking regions of mouse Zp-2 and Zp-3 genes and their human homologs contain five short DNA sequences (4 to 12 bp) that are 60 to 100% identical and are approximately equidistant upstream of the TATAA box in the four genes. Mutation of these five elements (I, IA, IIB, Ill, and IV) in Zp-luciferase constructs demonstrates that the 12-bp element IV, positioned approximately 200 bp upstream from the TATAA box, is necessary for high-level expression from the mouse Zp-2 and Zp-3 promoters after microinjection into the nuclei of 50-,um-diameter oocytes. Injection of mini'mal Zp-3 promoter constructs containing element IV in either 6197

Genetics, 1998

The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation i... more The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation in T2 disrupts notochord development, pointing to the existence of a second T/t complex gene involved in axis development. T2 encodes a novel protein that is disrupted by an insertion in T2(Bob) mice. Sequence analysis of T2 from several t haplotypes shows that they all share the same changed stop codon, and, thus, T2 is a candidate gene for the t complex tail interaction factor. T1, T2, and the unlinked t-int are distinct and unrelated loci, and mutations in these genes do not complement one another genetically. Either their products interact in the same pathway during the genesis of the embryonic axis, or the T/t region itself is truly complex.

Antiviral Therapy

Objectives To identify and characterize the siRNA duplexes that are effective for inhibition of S... more Objectives To identify and characterize the siRNA duplexes that are effective for inhibition of SARS-CoV infection and replication in the non-human primate cells. This in vitro study will serve as the foundation for development of novel anti-SARS therapeutics. Methods 48 siRNA sequences were designed for targeting regions throughout entire SARS-CoV genome RNA including open-reading frames for several key proteins. Chemically synthesized siRNA duplexes were transfected into foetal rhesus kidney (FRhK-4) cells prior to or after SARS-CoV infection. The inhibitory effects of the siRNAs were evaluated for reductions of intracellular viral genome copy number and viral titres in the cell culture medium measured by Q-RT-PCR and CPE-based titration, respectively. Four siRNA duplexes were found to achieve potent inhibition of SARS-CoV infection and replication. A prolonged prophylactic effect of siRNA duplexes with up to 90% inhibition that lasted for at least 72 h was observed. Combination o...

Developmental Biology, 1995

A new phenotype mapping to the t-complex, which is designated Brachyury the Second (T2), is chara... more A new phenotype mapping to the t-complex, which is designated Brachyury the Second (T2), is characterized by a slightly shortened tail in heterozygotes and homozygous failure to form an organized notochord with subsequent abnormal development of posterior somites and neural tube. The phenotype of T2 superficially resembles that of Brachyury; however, there are several important differences. Brachyury homozygotes fail to make posterior somites, notochord, floor plate, and a placental connection, resulting in death by 10.5 days of development. In contrast, T2 homozygotes make posterior somites, scattered notochord cells, and floorplate and achieve an allantoic connection. However, despite making a maternal connection, T2 homozygotes cease development at E11.5 and die soon after. We have cloned and analyzed the transgene insertion site, which maps within 100 kb of the Brachyury gene, but does not seem to physically interrupt nor affect transcription from that locus. The existence of a second gene mapping near Brachyury and affecting the same developmental processes was alluded to over 50 years ago and has been debated ever since. An embryological description of T2 is presented, as is a discussion of the implications of a single, larger Brachyury locus versus two closely linked genes coordinately regulating axial development.

Nucleic Acids Research, 2007

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) ... more Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (800 siRNAs, 400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.

The Journal of laboratory and clinical medicine, 1984

Antiserum to cobalamin was raised in rabbits by immunization with the monocarboxyl derivative of ... more Antiserum to cobalamin was raised in rabbits by immunization with the monocarboxyl derivative of cyanocobalamin coupled to albumin. The antiserum was treated to remove transcobalamin II and transcobalamin I. The partially purified antibody bound free cyano[57Co]cobalamin, but not the vitamin precoupled to the transcobalamins. Cyano[57Co]cobalamin bound by the antiserum eluted from Sephadex G-200 as a single peak with a mol wt of 160,000 and was precipitated by goat anti-rabbit gamma globulin, indicating that the vitamin was bound to an IgG immunoglobulin. Unlabeled cyanocobalamin and hydroxocobalamin competitively inhibited the binding of cyano[57Co]cobalamin to this antibody. Neither adenosylcobalamin, in a similar concentration range, nor cyanocobinamide at a concentration 30-fold greater than the tracer cobalamin competed appreciably with the binding of cyano[57Co] cobalamin. The association constant for the interaction of the antibody with cyanocobalamin and cyanocobinamide was ...

Genetics, 1998

The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation i... more The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation in T2 disrupts notochord development, pointing to the existence of a second T/t complex gene involved in axis development. T2 encodes a novel protein that is disrupted by an insertion in T2(Bob) mice. Sequence analysis of T2 from several t haplotypes shows that they all share the same changed stop codon, and, thus, T2 is a candidate gene for the t complex tail interaction factor. T1, T2, and the unlinked t-int are distinct and unrelated loci, and mutations in these genes do not complement one another genetically. Either their products interact in the same pathway during the genesis of the embryonic axis, or the T/t region itself is truly complex.

EMBO molecular medicine, Feb 18, 2015

There is an urgent need for new tools to combat the ongoing tuberculosis (TB) pandemic. Gene expr... more There is an urgent need for new tools to combat the ongoing tuberculosis (TB) pandemic. Gene expression profiles based on blood signatures have proved useful in identifying genes that enable classification of TB patients, but have thus far been complex. Using real-time PCR analysis, we evaluated the expression profiles from a large panel of genes in TB patients and healthy individuals in an Indian cohort. Classification models were built and validated for their capacity to discriminate samples from TB patients and controls within this cohort and on external independent gene expression datasets. A combination of only four genes distinguished TB patients from healthy individuals in both cross-validations and on separate validation datasets with very high accuracy. An external validation on two distinct cohorts using a real-time PCR setting confirmed the predictive power of this 4-gene tool reaching sensitivity scores of 88% with a specificity of around 75%. Moreover, this gene signatu...

Scientific reports, 2015

Our objective was to identify microRNA (miRNA) biomarkers of drug-induced liver and kidney injury... more Our objective was to identify microRNA (miRNA) biomarkers of drug-induced liver and kidney injury by profiling the circulating miRNome in patients with acetaminophen overdose. Plasma miRNAs were quantified in age- and sex-matched overdose patients with (N = 27) and without (N = 27) organ injury (APAP-TOX and APAP-no TOX, respectively). Classifier miRNAs were tested in a separate cohort (N = 81). miRNA specificity was determined in non-acetaminophen liver injury and murine models. Sensitivity was tested by stratification of patients at hospital presentation (N = 67). From 1809 miRNAs, 75 were 3-fold or more increased and 46 were 3-fold or more decreased with APAP-TOX. A 16 miRNA classifier model accurately diagnosed APAP-TOX in the test cohort. In humans, the miRNAs with the largest increase (miR-122-5p, miR-885-5p, miR-151a-3p) and the highest rank in the classifier model (miR-382-5p) accurately reported non-acetaminophen liver injury and were unaffected by kidney injury. miR-122-5p...

The Journal of laboratory and clinical medicine, 1984

Antiserum to cobalamin was raised in rabbits by immunization with the monocarboxyl derivative of ... more Antiserum to cobalamin was raised in rabbits by immunization with the monocarboxyl derivative of cyanocobalamin coupled to albumin. The antiserum was treated to remove transcobalamin II and transcobalamin I. The partially purified antibody bound free cyano[57Co]cobalamin, but not the vitamin precoupled to the transcobalamins. Cyano[57Co]cobalamin bound by the antiserum eluted from Sephadex G-200 as a single peak with a mol wt of 160,000 and was precipitated by goat anti-rabbit gamma globulin, indicating that the vitamin was bound to an IgG immunoglobulin. Unlabeled cyanocobalamin and hydroxocobalamin competitively inhibited the binding of cyano[57Co]cobalamin to this antibody. Neither adenosylcobalamin, in a similar concentration range, nor cyanocobinamide at a concentration 30-fold greater than the tracer cobalamin competed appreciably with the binding of cyano[57Co] cobalamin. The association constant for the interaction of the antibody with cyanocobalamin and cyanocobinamide was ...

The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T ). A mutation ... more The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T ). A mutation in T2 disrupts notochord development, pointing to the existence of a second T/t complex gene involved in axis development. T2 encodes a novel protein that is disrupted by an insertion in T2 Bob mice. Sequence analysis of T2 from several t haplotypes

The HZ5-feline sarcoma virus (FeSV) is a new acute transforming feline retrovirus which was isola... more The HZ5-feline sarcoma virus (FeSV) is a new acute transforming feline retrovirus which was isolated from a multicentric fibrosarcoma of a domestic cat. The HZ5-FeSV transforms fibroblasts in vitro and is replication defective. A biologically active integrated HZ5-FeSV provirus was molecularly cloned from cellular DNA of HZ5-FeSV-infected FRE-3A rat cells. The HZ5-FeSV has oncogene homology with the fms sequences of

Molecular and cellular biology, 1991

The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), p... more The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), performs crucial functions at fertilization and in early development. The transcripts encoding mouse ZP2 and ZP3 are coordinately expressed and accumulate in oocytes during a 2-week growth phase prior to ovulation. The 5'-flanking regions of mouse Zp-2 and Zp-3 genes and their human homologs contain five short DNA sequences (4 to 12 bp) that are 60 to 100% identical and are approximately equidistant upstream of the TATAA box in the four genes. Mutation of these five elements (I, IIA, IIB, III, and IV) in Zp-luciferase constructs demonstrates that the 12-bp element IV, positioned approximately 200 bp upstream from the TATAA box, is necessary for high-level expression from the mouse Zp-2 and Zp-3 promoters after microinjection into the nuclei of 50-microns-diameter oocytes. Injection of minimal Zp-3 promoter constructs containing element IV in either orientation also resulted in high le...

Oligonucleotides, 2009

Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that... more Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that generate highly functional siRNAs for gene knockdown. Nevertheless, "difficult-to-silence" target messenger RNAs (mRNAs) still exist for which no functionally validated siRNAs are available. MicroRNA (miRNA) sites in the mRNA 3'UTR, which interact with miRNA-loaded RNA-induced silencing complex (miRISC) for posttranscriptional gene regulation, provide alternative potentially accessible sites for siRNA. To investigate this, we designed siRNAs directed against single putative miRNA sites (misiRNAs) as predicted by miRNA target databases as well as siRNAs against other regions within the 3'UTR of "difficult-to-silence" targets in this proof-of-principle study. Although their design was not fully optimized, the misiRNAs generally caused higher knockdown than previously designed siRNAs for these targets. In general, knockdown by misiRNAs targeting the miRNA seed region was specific for the target mRNA, and misiRNAs targeting 1 nt upstream of miRNA seed region were similarly potent. We also systematically screened the 3'UTR of two mRNA targets using siRNA-tiling experiments. 5' and 3' regions of the p21-activated kinase 6 (PAK6) 3'UTR were found accessible, whereas the middle portion was largely inaccessible for siRNA knockdown. In ribosomal protein S6 kinase (RPS6KB1) 3'UTR, however, only the 5' region was accessible for siRNA knockdown. Detailed analysis of 10 further "difficult-to-silence" targets revealed that siRNA accessibility at the mRNA 3' end is not a general phenomenon, at least in "difficult-to-silence" targets, as we could not detect significant knockdown by siRNAs directed against this region.

…, 2009

Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that... more Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that generate highly functional siRNAs for gene knockdown. Nevertheless,“difficult-to-silence” target messenger RNAs (mRNAs) still exist for which no ...

Developmental Biology, 1993

Cancer Research, 2010

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We have used a nove... more Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We have used a novel real time PCR approach to profile global changes in microRNA expression during adipocytic differentiation of 3T3-L1 fibroblast cells. This profiling strategy makes use of a novel two stage screen resulting in a substantial increase in throughput and decrease in reagent use. In the first step, cDNA is prepared from individual samples of undifferentiated fibroblast, and cells at early and late stages of Adipocyte differentiation. A pooled cDNA sample prepared by pooling equimolar amounts of individual cDNAs was used in an initial profiling experiment to identify microRNAs that are expressed at detectable levels in the experimental system. Profiling was performed in triplicate with real time PCR assays for 591 mouse miRNAs (miRBase v12.0). In the second stage of screening, individual cDNA samples were each analyzed in triplicate using the assays for miRNAs that showed expression in the pooled cDNA sample. Expression data was normalized against a panel of small non-coding RNAs and was expressed as x-fold changes relative to expresion in undifferentiated 3T3L1 cells. Our results indicate that 192 (∼32%) miRNAs described in miRBase v12.0 were detectable in resting and differentiated 3T3-L1 cells, and of these, 38 (or 20%) showed differential regulation during adipogenesis (x-fold change>3.0). When compared to data from previous array-based profiling studies, our screen identified additional differentially regulated miRNAs. In addition to profiling changes in mature miRNA levels, miRNA precursor stem loop transcripts were also quantified to gain insight into post transcriptional regulation (if any) of miRNA biogenesis during adipocyte differentiation. The high specificity and sensitivity of the miScript RT-PCR system, together with a novel sample-pooling strategy makes this system a method of choice for miRNA expression profiling. Pooled primary profiling can substantially reduce the total number of PCR reactions which need to be performed and increases assay throughput. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3033.

Developmental biology, 1993

Molecular and cellular …, 1991

The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), p... more The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), performs crucial functions at fertilization and in early development. The transcripts encoding mouse ZP2 and ZP3 are coordinately expressed and accumulate in oocytes during a 2-week growth phase prior to ovulation. The 5'-flanking regions of mouse Zp-2 and Zp-3 genes and their human homologs contain five short DNA sequences (4 to 12 bp) that are 60 to 100% identical and are approximately equidistant upstream of the TATAA box in the four genes. Mutation of these five elements (I, IA, IIB, Ill, and IV) in Zp-luciferase constructs demonstrates that the 12-bp element IV, positioned approximately 200 bp upstream from the TATAA box, is necessary for high-level expression from the mouse Zp-2 and Zp-3 promoters after microinjection into the nuclei of 50-,um-diameter oocytes. Injection of mini'mal Zp-3 promoter constructs containing element IV in either 6197

Genetics, 1998

The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation i... more The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation in T2 disrupts notochord development, pointing to the existence of a second T/t complex gene involved in axis development. T2 encodes a novel protein that is disrupted by an insertion in T2(Bob) mice. Sequence analysis of T2 from several t haplotypes shows that they all share the same changed stop codon, and, thus, T2 is a candidate gene for the t complex tail interaction factor. T1, T2, and the unlinked t-int are distinct and unrelated loci, and mutations in these genes do not complement one another genetically. Either their products interact in the same pathway during the genesis of the embryonic axis, or the T/t region itself is truly complex.