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1.  Reciprocal knock-in mice to investigate the functional redundancy of lamin B1 and lamin B2 
Molecular Biology of the Cell  2014;25(10):1666-1675.
To assess the redundancy of lamins B1 and B2, knock-in lines were created that produce lamin B2 from the Lmnb1 locus and lamin B1 from the Lmnb2 locus. Both lines developed severe neurodevelopmental abnormalities, indicating that the abnormalities elicited by the loss of one B-type lamin cannot be prevented by increased synthesis of the other.
Lamins B1 and B2 (B-type lamins) have very similar sequences and are expressed ubiquitously. In addition, both Lmnb1- and Lmnb2-deficient mice die soon after birth with neuronal layering abnormalities in the cerebral cortex, a consequence of defective neuronal migration. The similarities in amino acid sequences, expression patterns, and knockout phenotypes raise the question of whether the two proteins have redundant functions. To investigate this topic, we generated “reciprocal knock-in mice”—mice that make lamin B2 from the Lmnb1 locus (Lmnb1B2/B2) and mice that make lamin B1 from the Lmnb2 locus (Lmnb2B1/B1). Lmnb1B2/B2 mice produced increased amounts of lamin B2 but no lamin B1; they died soon after birth with neuronal layering abnormalities in the cerebral cortex. However, the defects in Lmnb1B2/B2 mice were less severe than those in Lmnb1-knockout mice, indicating that increased amounts of lamin B2 partially ameliorate the abnormalities associated with lamin B1 deficiency. Similarly, increased amounts of lamin B1 in Lmnb2B1/B1 mice did not prevent the neurodevelopmental defects elicited by lamin B2 deficiency. We conclude that lamins B1 and B2 have unique roles in the developing brain and that increased production of one B-type lamin does not fully complement loss of the other.
doi:10.1091/mbc.E14-01-0683
PMCID: PMC4019497  PMID: 24672053
2.  Draft Genome Sequence of a Rare Smut Relative, Tilletiaria anomala UBC 951 
Genome Announcements  2014;2(3):e00539-14.
The draft genome sequence of the smut fungus Tilletiaria anomala UBC 951 (Basidiomycota, Ustilaginomycotina) is presented. The sequenced genome size is 18.7 Mb, consisting of 289 scaffolds and a total of 6,810 predicted genes. This is the first genome sequence published for a fungus in the order Georgefisheriales (Exobasidiomycetes).
doi:10.1128/genomeA.00539-14
PMCID: PMC4056295  PMID: 24926052
3.  Lipin-1 and lipin-3 together determine adiposity in vivo☆ 
Molecular Metabolism  2013;3(2):145-154.
The lipin protein family of phosphatidate phosphatases has an established role in triacylglycerol synthesis and storage. Physiological roles for lipin-1 and lipin-2 have been identified, but the role of lipin-3 has remained mysterious. Using lipin single- and double-knockout models we identified a cooperative relationship between lipin-3 and lipin-1 that influences adipogenesis in vitro and adiposity in vivo. Furthermore, natural genetic variations in Lpin1 and Lpin3 expression levels across 100 mouse strains correlate with adiposity. Analysis of PAP activity in additional metabolic tissues from lipin single- and double-knockout mice also revealed roles for lipin-1 and lipin-3 in spleen, kidney, and liver, for lipin-1 alone in heart and skeletal muscle, and for lipin-1 and lipin-2 in lung and brain. Our findings establish that lipin-1 and lipin-3 cooperate in vivo to determine adipose tissue PAP activity and adiposity, and may have implications in understanding the protection of lipin-1-deficient humans from overt lipodystrophy.
doi:10.1016/j.molmet.2013.11.008
PMCID: PMC3953701  PMID: 24634820
Gene family; Knockout mouse; Adipogenesis; Triacylglycerol; Glycerolipid biosynthesis
4.  Compromised Mitochondrial Fatty Acid Synthesis in Transgenic Mice Results in Defective Protein Lipoylation and Energy Disequilibrium 
PLoS ONE  2012;7(10):e47196.
A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor.
doi:10.1371/journal.pone.0047196
PMCID: PMC3471957  PMID: 23077570
5.  An absence of both lamin B1 and lamin B2 in keratinocytes has no effect on cell proliferation or the development of skin and hair 
Human Molecular Genetics  2011;20(18):3537-3544.
Nuclear lamins are usually classified as A-type (lamins A and C) or B-type (lamins B1 and B2). A-type lamins have been implicated in multiple genetic diseases but are not required for cell growth or development. In contrast, B-type lamins have been considered essential in eukaryotic cells, with crucial roles in DNA replication and in the formation of the mitotic spindle. Knocking down the genes for B-type lamins (LMNB1, LMNB2) in HeLa cells has been reported to cause apoptosis. In the current study, we created conditional knockout alleles for mouse Lmnb1 and Lmnb2, with the goal of testing the hypothesis that B-type lamins are crucial for the growth and viability of mammalian cells in vivo. Using the keratin 14-Cre transgene, we bred mice lacking the expression of both Lmnb1 and Lmnb2 in skin keratinocytes (Lmnb1Δ/ΔLmnb2Δ/Δ). Lmnb1 and Lmnb2 transcripts were absent in keratinocytes of Lmnb1Δ/ΔLmnb2Δ/Δ mice, and lamin B1 and lamin B2 proteins were undetectable. But despite an absence of B-type lamins in keratinocytes, the skin and hair of Lmnb1Δ/ΔLmnb2Δ/Δ mice developed normally and were free of histological abnormalities, even in 2-year-old mice. After an intraperitoneal injection of bromodeoxyuridine (BrdU), similar numbers of BrdU-positive keratinocytes were observed in the skin of wild-type and Lmnb1Δ/ΔLmnb2Δ/Δ mice. Lmnb1Δ/ΔLmnb2Δ/Δ keratinocytes did not exhibit aneuploidy, and their growth rate was normal in culture. These studies challenge the concept that B-type lamins are essential for proliferation and vitality of eukaryotic cells.
doi:10.1093/hmg/ddr266
PMCID: PMC3159554  PMID: 21659336
6.  The mammalian gene function resource: the international knockout mouse consortium 
Bradley, Allan | Anastassiadis, Konstantinos | Ayadi, Abdelkader | Battey, James F. | Bell, Cindy | Birling, Marie-Christine | Bottomley, Joanna | Brown, Steve D. | Bürger, Antje | Bult, Carol J. | Bushell, Wendy | Collins, Francis S. | Desaintes, Christian | Doe, Brendan | Economides, Aris | Eppig, Janan T. | Finnell, Richard H. | Fletcher, Colin | Fray, Martin | Frendewey, David | Friedel, Roland H. | Grosveld, Frank G. | Hansen, Jens | Hérault, Yann | Hicks, Geoffrey | Hörlein, Andreas | Houghton, Richard | Hrabé de Angelis, Martin | Huylebroeck, Danny | Iyer, Vivek | de Jong, Pieter J. | Kadin, James A. | Kaloff, Cornelia | Kennedy, Karen | Koutsourakis, Manousos | Kent Lloyd, K. C. | Marschall, Susan | Mason, Jeremy | McKerlie, Colin | McLeod, Michael P. | von Melchner, Harald | Moore, Mark | Mujica, Alejandro O. | Nagy, Andras | Nefedov, Mikhail | Nutter, Lauryl M. | Pavlovic, Guillaume | Peterson, Jane L. | Pollock, Jonathan | Ramirez-Solis, Ramiro | Rancourt, Derrick E. | Raspa, Marcello | Remacle, Jacques E. | Ringwald, Martin | Rosen, Barry | Rosenthal, Nadia | Rossant, Janet | Ruiz Noppinger, Patricia | Ryder, Ed | Schick, Joel Zupicich | Schnütgen, Frank | Schofield, Paul | Seisenberger, Claudia | Selloum, Mohammed | Simpson, Elizabeth M. | Skarnes, William C. | Smedley, Damian | Stanford, William L. | Francis Stewart, A. | Stone, Kevin | Swan, Kate | Tadepally, Hamsa | Teboul, Lydia | Tocchini-Valentini, Glauco P. | Valenzuela, David | West, Anthony P. | Yamamura, Ken-ichi | Yoshinaga, Yuko | Wurst, Wolfgang
Mammalian Genome  2012;23(9-10):580-586.
In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.
doi:10.1007/s00335-012-9422-2
PMCID: PMC3463800  PMID: 22968824
7.  A resource for the conditional ablation of microRNAs in the mouse 
Cell reports  2012;1(4):385-391.
Summary
The importance of miRNAs during development and disease processes is well established. However, most studies have been done in cells or with patient tissues, and therefore the physiological roles of miRNAs are not well understood. To unravel in vivo functions of miRNAs, we have generated conditional, reporter-tagged knockout-first mice for numerous evolutionarily conserved miRNAs. Here we report the generation of 162 miRNA targeting vectors, 64 targeted ES cell lines, and 46 germline-transmitted miRNA knockout mice. In vivo lacZ reporter analysis in 18 lines revealed highly tissue-specific expression patterns and their miRNA expression profiling matched closely with published expression data. Most miRNA knockout mice tested were viable, supporting a mechanism by which miRNAs act redundantly with other miRNAs or other pathways. These data and collection of resources will be of value for the in vivo dissection of miRNA functions in mouse models.
doi:10.1016/j.celrep.2012.02.008
PMCID: PMC3345170  PMID: 22570807
8.  Deficiencies in lamin B1 and lamin B2 cause neurodevelopmental defects and distinct nuclear shape abnormalities in neurons 
Molecular Biology of the Cell  2011;22(23):4683-4693.
Lamin B1 is essential for neuronal migration and progenitor proliferation during the development of the cerebral cortex. The observation of distinct phenotypes of Lmnb1- and Lmnb2-knockout mice and the differences in the nuclear morphology of cortical neurons in vivo suggest that lamin B1 and lamin B2 play distinct functions in the developing brain.
Neuronal migration is essential for the development of the mammalian brain. Here, we document severe defects in neuronal migration and reduced numbers of neurons in lamin B1–deficient mice. Lamin B1 deficiency resulted in striking abnormalities in the nuclear shape of cortical neurons; many neurons contained a solitary nuclear bleb and exhibited an asymmetric distribution of lamin B2. In contrast, lamin B2 deficiency led to increased numbers of neurons with elongated nuclei. We used conditional alleles for Lmnb1 and Lmnb2 to create forebrain-specific knockout mice. The forebrain-specific Lmnb1- and Lmnb2-knockout models had a small forebrain with disorganized layering of neurons and nuclear shape abnormalities, similar to abnormalities identified in the conventional knockout mice. A more severe phenotype, complete atrophy of the cortex, was observed in forebrain-specific Lmnb1/Lmnb2 double-knockout mice. This study demonstrates that both lamin B1 and lamin B2 are essential for brain development, with lamin B1 being required for the integrity of the nuclear lamina, and lamin B2 being important for resistance to nuclear elongation in neurons.
doi:10.1091/mbc.E11-06-0504
PMCID: PMC3226484  PMID: 21976703
9.  Comparative and demographic analysis of orangutan genomes 
Locke, Devin P. | Hillier, LaDeana W. | Warren, Wesley C. | Worley, Kim C. | Nazareth, Lynne V. | Muzny, Donna M. | Yang, Shiaw-Pyng | Wang, Zhengyuan | Chinwalla, Asif T. | Minx, Pat | Mitreva, Makedonka | Cook, Lisa | Delehaunty, Kim D. | Fronick, Catrina | Schmidt, Heather | Fulton, Lucinda A. | Fulton, Robert S. | Nelson, Joanne O. | Magrini, Vincent | Pohl, Craig | Graves, Tina A. | Markovic, Chris | Cree, Andy | Dinh, Huyen H. | Hume, Jennifer | Kovar, Christie L. | Fowler, Gerald R. | Lunter, Gerton | Meader, Stephen | Heger, Andreas | Ponting, Chris P. | Marques-Bonet, Tomas | Alkan, Can | Chen, Lin | Cheng, Ze | Kidd, Jeffrey M. | Eichler, Evan E. | White, Simon | Searle, Stephen | Vilella, Albert J. | Chen, Yuan | Flicek, Paul | Ma, Jian | Raney, Brian | Suh, Bernard | Burhans, Richard | Herrero, Javier | Haussler, David | Faria, Rui | Fernando, Olga | Darré, Fleur | Farré, Domènec | Gazave, Elodie | Oliva, Meritxell | Navarro, Arcadi | Roberto, Roberta | Capozzi, Oronzo | Archidiacono, Nicoletta | Valle, Giuliano Della | Purgato, Stefania | Rocchi, Mariano | Konkel, Miriam K. | Walker, Jerilyn A. | Ullmer, Brygg | Batzer, Mark A. | Smit, Arian F. A. | Hubley, Robert | Casola, Claudio | Schrider, Daniel R. | Hahn, Matthew W. | Quesada, Victor | Puente, Xose S. | Ordoñez, Gonzalo R. | López-Otín, Carlos | Vinar, Tomas | Brejova, Brona | Ratan, Aakrosh | Harris, Robert S. | Miller, Webb | Kosiol, Carolin | Lawson, Heather A. | Taliwal, Vikas | Martins, André L. | Siepel, Adam | RoyChoudhury, Arindam | Ma, Xin | Degenhardt, Jeremiah | Bustamante, Carlos D. | Gutenkunst, Ryan N. | Mailund, Thomas | Dutheil, Julien Y. | Hobolth, Asger | Schierup, Mikkel H. | Chemnick, Leona | Ryder, Oliver A. | Yoshinaga, Yuko | de Jong, Pieter J. | Weinstock, George M. | Rogers, Jeffrey | Mardis, Elaine R. | Gibbs, Richard A. | Wilson, Richard K.
Nature  2011;469(7331):529-533.
“Orangutan” is derived from the Malay term “man of the forest” and aptly describes the Southeast Asian great apes native to Sumatra and Borneo. The orangutan species, Pongo abelii (Sumatran) and Pongo pygmaeus (Bornean), are the most phylogenetically distant great apes from humans, thereby providing an informative perspective on hominid evolution. Here we present a Sumatran orangutan draft genome assembly and short read sequence data from five Sumatran and five Bornean orangutan genomes. Our analyses reveal that, compared to other primates, the orangutan genome has many unique features. Structural evolution of the orangutan genome has proceeded much more slowly than other great apes, evidenced by fewer rearrangements, less segmental duplication, a lower rate of gene family turnover and surprisingly quiescent Alu repeats, which have played a major role in restructuring other primate genomes. We also describe the first primate polymorphic neocentromere, found in both Pongo species, emphasizing the gradual evolution of orangutan genome structure. Orangutans have extremely low energy usage for a eutherian mammal1, far lower than their hominid relatives. Adding their genome to the repertoire of sequenced primates illuminates new signals of positive selection in several pathways including glycolipid metabolism. From the population perspective, both Pongo species are deeply diverse; however, Sumatran individuals possess greater diversity than their Bornean counterparts, and more species-specific variation. Our estimate of Bornean/Sumatran speciation time, 400k years ago (ya), is more recent than most previous studies and underscores the complexity of the orangutan speciation process. Despite a smaller modern census population size, the Sumatran effective population size (Ne) expanded exponentially relative to the ancestral Ne after the split, while Bornean Ne declined over the same period. Overall, the resources and analyses presented here offer new opportunities in evolutionary genomics, insights into hominid biology, and an extensive database of variation for conservation efforts.
doi:10.1038/nature09687
PMCID: PMC3060778  PMID: 21270892
10.  Targeted Disruption of the Idol Gene Alters Cellular Regulation of the Low-Density Lipoprotein Receptor by Sterols and Liver X Receptor Agonists ▿ §  
Molecular and Cellular Biology  2011;31(9):1885-1893.
Previously, we identified the E3 ubiquitin ligase Idol (inducible degrader of the low-density lipoprotein [LDL] receptor [LDLR]) as a posttranscriptional regulator of the LDLR pathway. Idol stimulates LDLR degradation through ubiquitination of its C-terminal domain, thereby limiting cholesterol uptake. Here we report the generation and characterization of mouse embryonic stem cells homozygous for a null mutation in the Idol gene. Cells lacking Idol exhibit markedly elevated levels of the LDLR protein and increased rates of LDL uptake. Furthermore, despite an intact sterol responsive element-binding protein (SREBP) pathway, Idol-null cells exhibit an altered response to multiple regulators of sterol metabolism, including serum, oxysterols, and synthetic liver X receptor (LXR) agonists. The ability of oxysterols and lipoprotein-containing serum to suppress LDLR protein levels is reduced, and the time course of suppression is delayed, in cells lacking Idol. LXR ligands have no effect on LDLR levels in Idol-null cells, indicating that Idol is required for LXR-dependent inhibition of the LDLR pathway. In line with these results, the half-life of the LDLR protein is prolonged in the absence of Idol. Finally, the ability of statins and PCSK9 to alter LDLR levels is independent of, and additive with, the LXR-Idol pathway. These results demonstrate that the LXR-Idol pathway is an important contributor to feedback inhibition of the LDLR by sterols and a biological determinant of cellular LDL uptake.
doi:10.1128/MCB.01469-10
PMCID: PMC3133228  PMID: 21343340
11.  Marking Embryonic Stem Cells with a 2A Self-Cleaving Peptide: A NKX2-5 Emerald GFP BAC Reporter 
PLoS ONE  2008;3(7):e2532.
Background
Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.
Methodology
Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.
Conclusions
Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library.
doi:10.1371/journal.pone.0002532
PMCID: PMC2430532  PMID: 18596956

Results 1-11 (11)