Frataxin knockin mouse

Friedreich ataxia is the consequence of frataxin deficiency, most often caused by a GAA repeat expansion in intron 1 of the corresponding gene. Frataxin is a mitochondrial protein involved in iron homeostasis. As an attempt to generate a mouse model of the disease, we introduced a (GAA)(230) repeat within the mouse frataxin gene by homologous recombination. GAA repeat knockin mice were crossed with frataxin knockout mice to obtain double heterozygous mice expressing 25-36% of wild-type frataxin levels. These mice were viable and did not develop anomalies of motor coordination, iron metabolism or response to iron loading. Repeats were meiotically and mitotically stable.     

The construction of transgenic and gene knockout/knockin mouse models of human disease

The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual’s gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care.     

Migraine prodromes separated from the aura: complete migraine.

Detailed questioning of 50 patients with uncomplicated migraine has shown that 17 had symptoms that preceded the headache phase by several hours. These prodromes consisted of changes in mood, behaviour, wakefulness, appetite, bowel activity, or fluid balance. The term "complete migraine" is proposed for attacks that include prodromal symptoms, whose occurrence implies an initial diffuse cerebral or hypothalamic disturbance.     

A rapid FACS-based strategy to isolate human gene knockin and knockout clones

Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.     

Progesterone involvement in breast development and tumorigenesis--as revealed by progesterone receptor "knockout" and "knockin" mouse models

In light of recent clinical trials, the debate concerning the risks and benefits of progestin-based postmenopausal hormone replacement therapy (HRT) has reached a renewed level of urgency. Irrespective of the position taken, the consensus is that more basic research needs to be performed to address progesterone's fundamental role in mammary development and tumorigenesis. Towards this end, the progesterone receptor knockout (PRKO) mouse demonstrated that progesterone is essential for pregnancy-associated mammary gland ductal side-branching and alveologenesis and that these morphological changes are dependent on progesterone-induced mammary epithelial proliferation. Importantly, the PRKO mouse showed that the progesterone-proliferative signal significantly contributes to mammary tumor susceptibility in an established mammary tumor model. Insight into the cellular mechanism(s) by which progesterone affects mammary morphogenesis has been disclosed by a new PR-LacZ knockin mouse, which revealed that PR's spatial expression pattern undergoes precise choreographed distributional changes that precede key stages in postnatal mammary development. In the case of early pregnancy, the segregation of cells undergoing progesterone-induced proliferation from those that express PR implicates a paracrine mode of action for progesterone-induced mammary epithelial proliferation, whereas the preparturient decline of PR expression underscores the need to remove this signal for full functional differentiation of this tissue. Our findings support the proposal that the mammary gland's normal response to the progesterone-signal is dependent upon specific spatial organizational patterns of PR expression and that derailment in these cellular processes may contribute to abnormal mammary development, including cancer. This review concludes by emphasizing the need to identify the downstream molecular targets that mediate progesterone's effects in this tissue. Identification of such targets will not only enhance our mechanistic understanding of progesterone's role in mammary development and cancer, but may also facilitate the formulation of new design strategies in breast cancer diagnosis and/or treatment.     

Outer hair cell-specific prestin-CreERT2 knockin mouse lines

Outer hair cells (OHCs) in the cochlea are crucial for the remarkable hearing sensitivity and frequency tuning. To understand OHC physiology and pathology, it is imperative to use mouse genetic tools to manipulate gene expression specifically in OHCs. Here, we generated two prestin knockin mouse lines: (1) the prestin-CreERT2 line, with an internal ribosome entry site-CreERT2-FRT-Neo-FRT cassette inserted into the prestin locus after the stop codon, and (2) the prestin-CreERT2-NN line, with the FRT-Neo-FRT removed subsequently. We characterized the inducible Cre activity of both lines by crossing them with the reporter lines CAG-eGFP and Ai6. Cre activity was induced with tamoxifen at various postnatal ages and only detected in OHCs, resembling the endogenous prestin expression pattern. Moreover, prestin-CreERT2+/-(heterozygotes) and +/+(homozygotes) as well as prestin-CreERT2-NN+/-mice displayed normal hearing. These two prestin-CreERT2 mouse lines are therefore useful tools to analyze gene function in OHCs in vivo.     

A Cacna1a knockin migraine mouse model with increased susceptibility to cortical spreading depression

Migraine is a common, disabling, multifactorial, episodic neurovascular disorder of unknown etiology. Familial hemiplegic migraine type 1 (FHM-1) is a Mendelian subtype of migraine with aura that is caused by missense mutations in the CACNA1A gene that encodes the alpha(1) subunit of neuronal Ca(v)2.1 Ca(2+) channels. We generated a knockin mouse model carrying the human pure FHM-1 R192Q mutation and found multiple gain-of-function effects. These include increased Ca(v)2.1 current density in cerebellar neurons, enhanced neurotransmission at the neuromuscular junction, and, in the intact animal, a reduced threshold and increased velocity of cortical spreading depression (CSD; the likely mechanism for the migraine aura). Our data show that the increased susceptibility for CSD and aura in migraine may be due to cortical hyperexcitability. The R192Q FHM-1 mouse is a promising animal model to study migraine mechanisms and treatments.     

Morphine-induced receptor endocytosis in a novel knockin mouse reduces tolerance and dependence

Opioid drugs, such as morphine, are among the most effective analgesics available. However, their utility for the treatment of chronic pain is limited by side effects including tolerance and dependence. Morphine acts primarily through the mu-opioid receptor (MOP-R) , which is also a target of endogenous opioids. However, unlike endogenous ligands, morphine fails to promote substantial receptor endocytosis both in vitro, and in vivo. Receptor endocytosis serves at least two important functions in signal transduction. First, desensitization and endocytosis act as an "off" switch by uncoupling receptors from G protein. Second, endocytosis functions as an "on" switch, resensitizing receptors by recycling them to the plasma membrane. Thus, both the off and on function of the MOP-R are altered in response to morphine compared to endogenous ligands. To examine whether the low degree of endocytosis induced by morphine contributes to tolerance and dependence, we generated a knockin mouse that expresses a mutant MOP-R that undergoes morphine-induced endocytosis. Morphine remains an excellent antinociceptive agent in these mice. Importantly, these mice display substantially reduced antinociceptive tolerance and physical dependence. These data suggest that opioid drugs with a pharmacological profile similar to morphine but the ability to promote endocytosis could provide analgesia while having a reduced liability for promoting tolerance and dependence.     

Improved methods for the generation of human gene knockout and knockin cell lines

Recent studies have demonstrated the utility of recombinant adeno-associated viral (rAAV) vectors in the generation of human knockout cell lines. The efficiency with which such cell lines can be generated using rAAV, in comparison with more extensively described plasmid-based approaches, has not been directly tested. In this report, we demonstrate that targeting constructs delivered by rAAV vectors were nearly 25-fold more efficient than transfected plasmids that target the same exon. In addition, we describe a novel vector configuration which we term the synthetic exon promoter trap (SEPT). This targeting element further improved the efficiency of knockout generation and uniquely facilitated the generation of knockin alterations. An rAAV-based SEPT targeting construct was used to transfer a mutant CTNNB1 allele, encoding an oncogenic form of β-catenin, from one cell line to another. This versatile method was thus shown to facilitate the efficient integration of small, defined sequence alterations into the chromosomes of cultured human cells.     

One-step knockin for inducible expression in mouse embryonic stem cells

Transgenesis enables the elucidation of gene function; however, constant transgene expression is not always desired. The tetracycline responsive system was devised to turn on and off transgene expression at will. It has two components: a doxycycline (dox)-controlled transactivator (TA) and an inducible expression cassette. Integration of these transgenes requires two transfection steps usually accomplished by sequential random integration. Unfortunately, random integration can be problematic due to chromatin position effects, integration of variable transgene units, and mutation at the integration site. Therefore, targeted transgenesis and knockin were developed to target the TA and the inducible expression cassette to a specific location, but these approaches can be costly in time, labor, and money. Here, we describe a one-step Cre-mediated knockin system in mouse embryonic stem cells that positions the TA and inducible expression cassette to a single location. Using this system, we show dox-dependent regulation of eGFP at the DNA topoisomerase 3β promoter. Because Cre-mediated recombination is used in lieu of gene targeting, this system is fast and efficient.     

Generation and analyses of R8L barttin knockin mouse

Barttin, a gene product of BSND, is one of four genes responsible for Bartter syndrome. Coexpression of barttin with ClC-K chloride channels dramatically induces the expression of ClC-K current via insertion of ClC-K-barttin complexes into plasma membranes. We previously showed that stably expressed R8L barttin, a disease-causing missense mutant, is retained in the endoplasmic reticulum (ER) of Madin-Darby canine kidney (MDCK) cells, with the barttin β-subunit remaining bound to ClC-K α-subunits (Hayama A, Rai T, Sasaki S, Uchida S. Histochem Cell Biol 119: 485-493, 2003). However, transient expression of R8L barttin in MDCK cells was reported to impair ClC-K channel function without affecting its subcellular localization. To investigate the pathogenesis in vivo, we generated a knockin mouse model of Bartter syndrome that carries the R8L mutation. These mice display disease-like phenotypes (hypokalemia, metabolic alkalosis, and decreased NaCl reabsorption in distal tubules) under a low-salt diet. Immunofluorescence and immunoelectron microscopy revealed that the plasma membrane localization of both R8L barttin and the ClC-K channel was impaired in these mice, and transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) as well as thiazide-sensitive chloride clearance were significantly reduced. This reduction in transepithelial chloride transport in tAL, which is totally dependent on ClC-K1/barttin, correlated well with the reduction in the amount of R8L barttin localized to plasma membranes. These results suggest that the major cause of Bartter syndrome type IV caused by R8L barttin mutation is its aberrant intracellular localization.     

Of mice and MEN1: Insulinomas in a conditional mouse knockout

Patients with multiple endocrine neoplasia type 1 (MEN1) develop multiple endocrine tumors, primarily affecting the parathyroid, pituitary, and endocrine pancreas, due to the inactivation of the MEN1 gene. A conditional mouse model was developed to evaluate the loss of the mouse homolog, Men1, in the pancreatic beta cell. Men1 in these mice contains exons 3 to 8 flanked by loxP sites, such that, when the mice are crossed to transgenic mice expressing cre from the rat insulin promoter (RIP-cre), exons 3 to 8 are deleted in beta cells. By 60 weeks of age, >80% of mice homozygous for the floxed Men1 gene and expressing RIP-cre develop multiple pancreatic islet adenomas. The formation of adenomas results in elevated serum insulin levels and decreased blood glucose levels. The delay in tumor appearance, even with early loss of both copies of Men1, implies that additional somatic events are required for adenoma formation in beta cells. Comparative genomic hybridization of beta cell tumor DNA from these mice reveals duplication of chromosome 11, potentially revealing regions of interest with respect to tumorigenesis.     

Modeling premature aging syndromes with the telomerase knockout mouse

Understanding the molecular basis of the aging process is a daunting problem, given the complex genetic and physiological changes that underlie human aging and the lack of genetically amenable primate model systems. However, analysis of mouse models exhibiting aging phenotypes reminiscent of those observed in elderly humans has enhanced our understanding of the biological mechanisms underlying mammalian aging. In particular, these mouse models have brought to light the importance of the DNA damage pathway during the aging process. Increased genomic instability is associated with accelerated cellular decline and manifestation of premature aging phenotypes in mice. Here I discuss how one form of genomic instability, initiated by critically short telomeres in the telomerase knockout mouse, perturb normal mammalian aging. Insights into the molecular pathways of the aging process may offer unprecedented opportunities to delay the deleterious effects of the aging process.     

The butyrylcholinesterase knockout mouse is obese on a high-fat diet

Butyrylcholinesterase (BChE) inactivates the appetite stimulating hormone octanoyl-ghrelin. The hypothesis was tested that BChE−/− mice would have abnormally high body weight and high levels of octanoyl-ghrelin. It was found that BChE−/− mice fed a standard 5% fat diet had normal body weight. However, BChE−/− mice fed a diet containing 11% fat became obese. Their obesity was not explained by increased levels of octanoyl-ghrelin, or by increased caloric intake, or by decreased exercise. Instead, a role for BChE in fat utilization was suggested.     

The eIF2A knockout mouse

Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the early 1970s as a factor capable of stimulating initiator methionyl-tRNAi (Met-tRNA^Met_i) binding to 40S ribosomal subunits in vitro. However, in contrast to the eIF2, which stimulates Met-tRNA^Met_i binding to 40S ribosomal subunits in a GTP-dependent manner, eIF2A didn't reveal any GTP-dependence, but instead was found to direct binding of the Met-tRNA^Met_i to 40S ribosomal subunits in a codon-dependent manner. eIF2A appears to be highly conserved across eukaryotic species, suggesting conservation of function in evolution. The yeast Saccharomyces cerevisae eIF2A null mutant revealed no apparent phenotype, however, it was found that in yeast eIF2A functions as a suppressor of internal ribosome entry site (IRES)-mediated translation. It was thus suggested that eIF2A my act by impinging on the expression of specific mRNAs. Subsequent studies in mammalian cell systems implicated eIF2A in non-canonical (non-AUG-dependent) translation initiation events involving near cognate UUG and CUG codons. Yet, the role of eIF2A in cellular functions remains largely enigmatic. As a first step toward characterization of the eIF2A function in mammalian systems in vivo, we have obtained homozygous eIF2A-total knockout (KO) mice, in which a gene trap cassette was inserted between eIF2A exons 1 and 2 disrupting expression of all exons downstream of the insertion. The KO mice strain is viable and to date displays no apparent phenotype. We believe that the eIF2A KO mice strain will serve as a valuable tool for researchers studying non-canonical initiation of translation in vivo.