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PubMed1997

A ribozyme-mediated, gene "knockdown" strategy for the identification of gene function in zebrafish.

The zebrafish system offers many unique opportunities for the study of molecular biology. To date, only random mutagenesis, and not directed gene knockouts, have been demonstrated in this system. To more fully develop the potential of the zebrafish system, an approach to effectively inhibit the expression of any targeted gene in the developing zebrafish embryo has been developed. This approach uses a transient, cytoplasmic, T7 expression system, injected into the fertilized zebrafish egg to rapidly produce high levels of a ribozyme directed against the mRNA encoded by the targeted gene to inhibit its expression. In a demonstration of this strategy, expression of the recessive dominant zebrafish no tail gene was effectively inhibited by using this strategy to yield a phenotype identical to that resulting from a known defective mutation in this same gene. This, ribozyme-mediated, message deletion strategy may have use in determining the function of genetic coding sequences of unknown function.

Animals↗
PubMed2006

Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening.

The ability to generate null mutants is essential for studying gene function. Gene knockouts in Caenorhabditis elegans can be generated in a high throughput manner using chemical mutagenesis followed by polymerase chain reaction (PCR) assays to detect deletions in a gene of interest. However, current methods for identifying deletions are time and labor intensive and are unable to efficiently detect small deletions. In this study, we expanded the method pioneered by Wei et al., which used the thermostable restriction enzyme PspGI and tested the usefulness of other thermostable restriction enzymes including BstUI, Tsp45I, ApeKI, and TfiI. We designed primers to flank one or multiple thermostable restriction enzymes sites in the genes of interest. The use of multiple enzymes and the optimization of PCR primer design enabled us to isolate deletion in 66.7% of the genes screened. The size of the deletions varied from 330 bp to 1 kb. This method should make it possible for small academic laboratories to rapidly isolate deletions in their genes of interest.

Alkylating Agents↗
PubMed2003

Exploring the overproduction of amino acids using the bilevel optimization framework OptKnock.

In this study, we modify and extend the bilevel optimization framework OptKnock for identifying gene knockout strategies in the Escherichia coli metabolic network, leading to the overproduction of representative amino acids and key precursors for all five families. These strategies span not only the central metabolic network genes but also the amino acid biosynthetic and degradation pathways. In addition to gene deletions, the transport rates of carbon dioxide, ammonia, and oxygen, as well as the secretion pathways for key metabolites, are introduced as optimization variables in the framework. Computational results demonstrate the importance of manipulating energy-producing/consuming pathways, controlling the uptake of nitrogen and oxygen, and blocking the secretion pathways of key competing metabolites. The identified pathway modifications include not only straightforward elimination of competing reactions but also a number of nonintuitive knockouts quite distant from the amino acid-producing pathways. Specifically, OptKnock suggests three reactions (i.e., pyruvate kinase, phosphotransacetylase, and ATPase) for deletion, in addition to the straightforward elimination of 2-ketoglutarate dehydrogenase, to generate a glutamate-overproducing mutant. Similarly, phosphofructokinase and ATPase are identified as promising knockout targets to complement the removal of pyruvate formate lyase and pyruvate dehydrogenase for enhancing the yield of alanine. Although OptKnock in its present form does not consider regulatory constraints, it does provide useful suggestions largely in agreement with existing practices and, more importantly, introduces a framework for incorporating additional modeling refinements as they become available.

Algorithms↗
PubMed2001

Targeted gene mutation approaches to the study of anxiety-like behavior in mice.

Studying the behavioral phenotypes of transgenic and gene knockout mice is a powerful means to better understand the pathophysiology of neuropsychiatric disorders and ultimately improve their treatment. This paper provides an overview of the methods and findings of studies that have tested for anxiety-related behavioral phenotypes in gene mutant mice. In the context of improving the side effect burden of benzodiazepines, gene targeting has been valuable for dissociating the functional roles (i.e., anxiolytic, sedative, amnestic) of individual GABA(A) receptor subunits. Supporting the link between abnormalities in CRH function and anxiety, CRH overexpressing transgenic mice and CRH-R2 receptor knockout mutants have displayed significantly increased anxiety-like behavior, while CRH-R1 receptor knockout mice have shown an anxiolytic-like phenotype. Consistent with an important role for the serotonergic system in anxiety, 5-HT1(A) receptor deficient mice have consistently exhibited heightened anxiety-like behavior, while the evidence from 5-HT1(B) and 5-HT2(C) deficient mice remains somewhat equivocal. Mutant mice lacking either of the monoamine degrading enzymes, MAOA or COMT, have shown a number of behavioral and neurological effects, including alterations in anxiety-like behavior. With enhanced spatial and temporal control over gene mutations, in combination with an improved battery of behavioral tests, gene mutant mice will provide an increasingly valuable tool for understanding the neural substrates of anxiety.

Animals↗
PubMed2002

Distinct involvement of the Jun-N-terminal kinase and NF-kappaB pathways in the repression of the human COL1A2 gene by TNF-alpha.

We used a gene knockout approach to elucidate the specific roles played by the Jun-N-terminal kinase (JNK) and NF-kappaB pathways downstream of TNF-alpha in the context of alpha(2) type I collagen gene (COL1A2) expression. In JNK1-/--JNK2-/- (JNK-/-) fibroblasts, TNF-alpha inhibited basal COL1A2 expression but had no effect on TGF-beta-driven gene transactivation unless jnk1 was introduced ectopically. Conversely, in NF-kappaB essential modulator-/- (NEMO-/-) fibroblasts, lack of NF-kappaB activation did not influence the antagonism exerted by TNF-alpha against TGF-beta but prevented repression of basal COL1A2 gene expression. Similar regulatory mechanisms take place in dermal fibroblasts, as evidenced using transfected dominant-negative forms of MKK4 and IKK-alpha, critical kinases upstream of the JNK and NF-kappaB pathways, respectively. These results represent the first demonstration of an alternate usage of distinct signaling pathways by TNF-alpha to inhibit the expression of a given gene, COL1A2, depending on its activation state.

Animals↗
PubMed2004

Identification of genes involved in VEGF-mediated vascular morphogenesis using embryonic stem cell-derived cystic embryoid bodies.

The vasculature forms during development via two processes, vasculogenesis and angiogenesis, in which vessels form de novo from angioblast precursors or as sprouts from pre-existing vessels, respectively. A common and critical aspect of both processes is vascular morphogenesis, which includes branching of endothelial cell cords and lumen formation. Although ample evidence support the central role of vascular endothelial growth factor (VEGF) in both vasculogenesis and angiogenesis, the role of VEGF in vascular morphogenesis is unclear and little is known about the regulation of vascular morphogenesis, in general. We have used the in vitro vessel differentiation system of embryonic stem (ES) cell-derived cystic embryonic bodies (CEB) as a model for studying VEGF-mediated vessel formation. Whereas CEB formed from wild-type ES cells make well-formed vessel-like structures, CEB derived from VEGF-null ES cells contain PECAM-1-positive endothelial cells, but these cells do not participate in vascular morphogenesis. Using gene expression microarray analysis to compare gene expression in these two systems, we have been able to identify many genes and novel ESTs that are downstream of VEGF function, and which may be involved in VEGF-mediated vascular morphogenesis including caveolin-1 and HEY-1. These results support using the CEB model, in combination with gene knockout ES cells, for studying vascular morphogenesis.

Animals↗
PubMed2006

Muscarinic receptors are affected by corticotropin-releasing hormone and c-fos gene disruptions: is there a mutual connection to adrenoceptors?

In the last decade, progress in gene disruption technology has allowed the study of the effects of the single-gene knockout (KO) on different molecules involved in the signaling cascade activated via muscarinic receptors. Many KO mice targeting muscarinic receptors have been developed, that is, all (M1-M5) muscarinic receptor KO mice (Wess, 2003) and acetylcholinesterase (AChE) KO mice(Xie et al., 2000). Recently, we have shown that these (AChE-/-) mice not only reveal changes in the number of muscarinic receptors in the heart, lung, cortex, and cerebellum but also in the number of adrenoceptors (Teplicky et al., 2004). Next, we studied whether the disruption of corticotropin-releasing hormone (CRH) or c-Fos could affect the properties of muscarinic receptors and adrenoceptors in the lungs and hearts of mice. The effects of immobilization stress in CRH KO animals were also studied.

Animals↗
PubMed2004

Homozygous deletion of early growth response 1 gene and critical limb ischemia after vascular ligation in mice: evidence for a central role in vascular homeostasis.

BACKGROUND: The early growth response 1 gene (Egr1) encodes for an immediate to early response transcription factor that is upregulated by changes in vascular strain and hypoxia and in turn upregulates the downstream expressions of a number of angiogenic growth factors. We therefore hypothesized that early growth response 1 may be a critical early messenger governing revascularization in the setting of acute vascular occlusions. METHODS: C57 BL/6 mice deficient in the Egr1 gene (knockout) and their wild-type litter mates underwent ligation and excision of the femoral artery with or without the previous administration of 2.7 x 10(9) particle units of an adenoviral vector coding for the vascular endothelial growth factor gene (VEGF) or Egr1. Distal hind limb perfusion was serially measured in these animals with laser Doppler perfusion imaging. RESULTS: Wild-type mice (n = 9) had nearly complete restitution of hind limb perfusion by day 35 after ligation. In contrast, all noninjected Egr1 knockout mice (n = 5) had severe ipsilateral limb necrosis develop between 1 and 4 days after ligation (P <.0001). Egr1 knockout mice injected with VEGF vector (n = 4) demonstrated significantly improved perfusion relative to baseline by postligation day 28, which persisted to postligation day 35 (P <.05). Egr1 knockout animals injected with Egr1 vector (n = 7) demonstrated a partial recovery of hind limb perfusion relative to VEGF vector-treated knockout animals at postligation day 4 (P <.01), which persisted to day 35. CONCLUSIONS: These findings suggest that early growth response 1 plays a pivotal role in reperfusion responses to vascular occlusion in mice and possibly other mammals.

Adenoviridae↗
PubMed2005

Genes encoding catalytic subunits of protein kinase A and risk of spina bifida.

BACKGROUND: PRKACA and PRKACB are genes encoding the cAMP-dependent protein kinase A (PKA) catalytic subunits alpha and beta, respectively. PKA is known to be involved in embryonic development, as it down-regulates the Hedgehog (Hh) signaling pathway, which is critical to normal pattern formation and morphogenesis. The PKA-deficient mouse model, which has only a single catalytic subunit, provided intriguing evidence demonstrating a relationship between decreased PKA activity and risk for posterior neural tube defects (NTDs) in the thoracic to sacral regions of gene-knockout mice. Unlike most other mutant mouse models of NTDs, the PKA-deficient mice develop spina bifida with 100% penetrance. We hypothesized that sequence variations in human genes encoding the catalytic subunits may alter the PKA activity and similarly increase the risk of spina bifida. METHODS: We sequenced the coding regions and the exon/intron boundaries of PRKACA and PRKACB. We also examined 3 common single-nucleotide polymorphisms (SNPs) of these 2 genes by allele discrimination. RESULTS: Five sequence variants in coding region and 2 intronic sequence variants proximal to exons were detected. None of the 3 SNPs examined in the association study appeared to be associated with substantially increased risk for spina bifida. CONCLUSIONS: Our results did not reveal a strong association between these PKA SNPs and spina bifida risk. Nonetheless, it is important to examine the possible gene-gene interactions between PRKACA and PRKACB when evaluating the risk for NTDs, as well as genes encoding regulatory subunits of PKA. In addition, interactions with other genes such as Sonic Hedgehog (SHH) should also be considered for future investigations.

Alleles↗
PubMed2002

Myostatin knockout in mice increases myogenesis and decreases adipogenesis.

Growth differentiation factor-8 (GDF-8), or Myostatin, plays an important inhibitory role during muscle development. Since muscle and adipose tissue develop from the same mesenchymal stem cells, we hypothesized that Myostatin gene knockout may cause a switch between myogenesis and adipogenesis. Male and female wild type (WT) and Myostatin knockout (KO) mice were sacrificed at 4, 8, and 12 weeks of age. The gluteus muscle (GM) was larger in KO mice compared to WT mice at 8 (P < 0.01) and 12 (P < 0.001) weeks. At 12 weeks, KO mice had decreased fat depots (P < 0.01). Compared to 12-week-old WT mice, serum leptin concentration in KO mice was lower (P < 0.001) and leptin mRNA expression was decreased (P < 0.01) in inguinal adipose tissue. CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) levels in adipose tissue were significantly lower in KO mice compared to WT mice. Thus, increased muscle development in Myostatin knockout mice is associated with reduced adipogenesis and consequently, decreased leptin secretion.

Adipose Tissue↗
PubMed2003

Human wild-type SEDL protein functionally complements yeast Trs20p but some naturally occurring SEDL mutants do not.

X-linked spondyloepiphyseal dysplasia tarda (SEDT, or SEDL) is a primary skeletal dysplasia affecting mostly spinal vertebral bodies and epiphyses. Previously, we have identified the SEDL gene and determined the spectrum of 21 different SEDL causing mutations. The SEDL gene is a highly conserved gene with an as yet unknown function. The yeast SEDL protein ortholog, Trs20p, has been isolated as a member of a large multi-protein complex ( approximately 10 proteins) called transport protein particle (TRAPP), which is involved in endoplasmic reticulum (ER)-to-Golgi transport. While the SEDL gene mutations cause a tissue-specific (epiphyses) and relatively mild phenotype, the Trs20p function is essential for the yeast cell. We now provide evidence that recombinant human SEDL protein is able to functionally complement the Saccharomyces cerevisiae TRS20 (TRAPP subunit 20 gene) knockout mutant. This finding strongly supports the speculated conserved nature of the SEDL/Trs20p function. To shed further light on the SEDL/Trs20p protein function, five different naturally occurring SEDL gene mutations have been tested in complementation studies. While two truncation mutations (157delAT and C271T) and one missense mutation (G139T) were unable to rescue the trs20Delta lethal phenotype, two other missense mutations (C218T and T389A) did complement trs20Delta. Interestingly, there is no obvious correlation between the nature and position of the SEDL mutation and the clinical severity of the disorder among the human SEDL patients. Although the identification of complementing SEDL gene mutations may suggest the existence of subtle phenotypic differences among SEDL patients, it might also point towards the identification of SEDL protein residues/domains specific for normal, vertebrate bone growth.

Amino Acid Sequence↗
PubMed2001

Transgenic knockouts as part of high-throughput, evidence-based target selection and validation strategies.

The worldwide genome sequencing projects are helping to define the size and complexity of the expressed genome and are thereby identifying an unprecedented number of genes of uncertain disease alignment and unknown function. It is widely recognized that, within the pharmaceutical industry, a significant commercial advantage will accrue to those companies that most effectively gather and integrate additional biological information into their therapeutic target selection and drug progression strategies. This article presents the rationale for including comparative phenotypic information obtained from transgenic gene knockouts as an integral part of any future therapeutic target selection strategy.

Journal Article↗
PubMed2003

Disruption of basal JNK activity differentially affects key fibroblast functions important for wound healing.

We used both a gene knockout approach and pharmacologic modulation to study the implication of the JNK pathway in regulating fibroblast motility, capacity to contract mechanically unloaded collagen gels, and type I collagen gene expression in vitro. These parameters, which are important for tissue repair, are positively regulated by transforming growth factor (TGF)-beta, a cytokine viewed as playing a master role during wound healing. We demonstrate that basal JNK activity is critical for fibroblast motility because (a) mouse embryo jnk-/- fibroblasts exhibit significantly lower ability to close mechanically induced cell layer wounds than their wild-type (wt) counterparts, and (b) wound closure by human dermal fibroblasts is dramatically impaired by the specific JNK inhibitor SP600125. junAA fibroblasts, in which amino acids Ser63 and Ser73 of c-Jun are replaced by two Ala residues so that c-Jun cannot be phosphorylated by JNK, also exhibited impaired motility, suggesting that c-Jun phosphorylation by JNK is critical for fibroblast migration. In sharp contrast to their lesser motility on plastic, jnk-/- and junAA fibroblasts contracted free-floating, mechanically unloaded, collagen lattices markedly faster than wt fibroblasts. Furthermore, basal mRNA steady-state levels for types I and III collagen genes were similar in jnk-/- and wt fibroblasts. Likewise, overexpression of a dominant-negative mutant form of MKK4 in dermal fibroblasts did not affect collagen expression. We also demonstrate that basal JNK activity does not affect either TGF-beta-induced collagen gene expression or lattice contraction, whereas on the other hand, the blockage of motility initiated by JNK inhibition cannot be overcome by TGF-beta. Together these results demonstrate discrete, yet significant and highly specific, regulation of fibroblast functions important for wound healing by basal JNK activity.

Cell Movement↗
PubMed1998

Alterations in dynamic heart rate control in the beta 1-adrenergic receptor knockout mouse.

beta 1-Adrenergic receptors (beta 1-ARs) are key targets of sympathetic nervous system activity and play a major role in the beat-to-beat regulation of cardiac chronotropy and inotropy. We employed a beta 1-AR gene knockout model to test the hypothesis that beta 1-AR function is critical for maintenance of resting heart rate and baroreflex responsiveness and, on the basis of its important role in regulating chronotropy and inotropy, is also required for maximal exercise capacity. Using an awake unrestrained mouse model, we demonstrate that resting heart rate and blood pressure are normal in beta 1-AR knockouts and that the qualitative responses to baroreflex stimulation are intact. Chronotropic reserve in beta 1-AR knockouts is markedly limited, with peak heart rates approximately 200 beats/min less than wild types. During graded treadmill exercise, heart rate is significantly depressed in beta 1-AR knockouts at all work loads, but despite this limitation, there are no reductions in maximal exercise capacity or metabolic indexes. Thus, in mice, the beta 1-AR is not essential for either maintenance of resting heart rate or for maximally stressed cardiovascular performance.

Adrenergic beta-Agonists↗
PubMed2003

Extracellular mediators in atherosclerosis and thrombosis: lessons from thrombin receptor knockout mice.

It is well appreciated that thrombin as well as other proteases can act as signaling molecules that specifically regulate cells by cleaving and activating members of a novel class of protease-activated receptors (PARs). The utility of gene knockout strategies to define and better comprehend the physiological role of specific proteins is perhaps best exemplified in the field of thrombin receptors. The development of PAR knockout mice has provided the unique opportunity to identify and characterize new members of this novel family of GPCRs, evaluate the interaction of PARs jointly expressed in common cells and tissues, and better understand the role of PARs in thrombosis, restenosis, vascular remodeling, angiogenesis, and inflammation. Presently, 4 members of the PAR family have been cloned and identified. In this review, we examine experimental evidence gleaned from PAR-/- mouse models as well as how the use of PAR-/- mice has provided insights toward understanding the physiological role of thrombin in cells of the vascular system and vascular pathology.

Animals↗
PubMed2006

Multiple shRNAs expressed by an inducible pol II promoter can knock down the expression of multiple target genes.

RNA interference (RNAi) has been increasingly used for reverse genetics. Both pol III and pol II promoters have been used to synthesize short hairpin RNA (shRNA) for knockdown of gene expression in mammalian cells and animals. Compared with gene knockout approaches, RNAi has the advantage of being simple, quick, and low cost. Here we describe a method that enhances this advantage where knockdown of expression of multiple genes in the same cells is required. A tetracycline-regulated pol II promoter construct allows the expression of up to three shRNA genes that have been cloned into introns of a transcript bearing green fluorescent protein (GFP) coding sequences. This method may be used to establish stable knockdown cell lines and may also prove useful for investigating gene-gene interactions in transgenic animals.

Animals↗
PubMed1996

Interleukin-1 involvement in the induction of leukemia inhibitory factor mRNA expression following axotomy of sympathetic ganglia.

Axotomy of superior cervical (sympathetic) ganglia (SCG) results in increased neuropeptide gene expression. In vitro, neuropeptide gene expression is similarly increased by exposure to the inflammatory cytokine interleukin-1 (IL-1). The effect of IL-1 in-vitro has been shown to be mediated by leukemia inhibitory factor (LIF). Since IL-1 regulates neuropeptide expression via LIF in vitro, we asked whether axotomy in vivo produces an increase in LIF mRNA, and whether that increase is regulated by IL-1 activity. Within 6 h following axotomy, ganglionic LIF mRNA is substantially elevated. Moreover, axotomy produces a rapid and transient increase in intraganglionic IL-1 beta mRNA, followed rapidly by an increase in ICAM-1 mRNA, thereby suggesting a local source of IL-1 activity. Pretreatment with the anti-inflammatory agent dexamethasone (DEX) reduces the increases of both IL-1 beta and LIF mRNAs following axotomy. mRNA encoding the specific signal-transducing Type I IL-1 receptor is present in unlesioned SCG in vivo, and increases following axotomy. Local application of IL-1 beta in vivo induces LIF mRNA even in uninjured ganglia, though not to the extent seen with axotomy. DEX treatment blocks this IL-1 beta-mediated increase in LIF mRNA. Therefore, DEX blocks the induction of LIF mRNA by inhibiting both the production of IL-1 and its action on LIF gene expression. Axotomy of a homozygous IL-1 receptor type I gene knockout mouse leads to a delayed and/or diminished induction of LIF mRNA in SCG, but does not prevent LIF mRNA expression. We conclude that while IL-1 is likely to be involved in the cascade of gene expression that follows axotomy, it alone is not sufficient to mediate the full induction of LIF mRNA by axotomy.

Animals↗
PubMed2006

CITED1 homozygous null mice display aberrant pubertal mammary ductal morphogenesis.

Expression microarray analysis identified CITED1 among a group of genes specifically upregulated in the pubertal mouse mammary gland. At puberty, CITED1 localizes to the luminal epithelial cell population of the mammary ducts and the body cells of the terminal end buds. Generation of CITED1 gene knockout mice showed that homozygous null mutants exhibit retarded mammary ductal growth at puberty and, in addition, dilated ductal structures with a lack of spatial restriction of the subtending branches. Analysis of CITED1 homozygous null and heterozygous null mammary gland gene expression using microarrays suggested that the mammary-specific phenotype seen in the homozygous null females is due to a disturbance in the transcription of a number of key mediators of pubertal ductal morphogenesis. These include estrogen and TGFbeta responsive genes, such as the EGFR/ErbB2 ligand, amphiregulin, whose transcription we suggest is directly or indirectly regulated by CITED1.

Animals↗