Highly interactive nature of flower‐specific enhancers and promoters,and its potential impact on tissue‐specific expression and engineering of multiple genes or agronomic traits

Molecular stacking enables multiple traits to be effectively engineered in crops using a single vector. However, the co‐existence of distinct plant promoters in the same transgenic unit might, like their mammalian counterparts, interfere with one another. In this study, we devised a novel approach to investigate enhancer–promoter and promoter–promoter interactions in transgenic plants and demonstrated that three of four flower‐specific enhancer/promoters were capable of distantly activating a pollen‐ and stigma‐specific Pps promoter (fused to the cytotoxic DT‐A gene) in other tissues, as revealed by novel tissue ablation phenotypes in transgenic plants. The NtAGI1 enhancer exclusively activated stamen‐ and carpel‐specific DT‐A expression, thus resulting in tissue ablation in an orientation‐independent manner; this activation was completely abolished by the insertion of an enhancer‐blocking insulator (EXOB) between the NtAGI1 enhancer and Pps promoter. Similarly, AGL8 and AP1Lb1, but not AP1La, promoters also activated distinct tissue‐specific DT‐A expression and ablation, with the former causing global growth retardation and the latter ablating apical inflorescences. While the tissue specificity of the enhancer/promoters generally defined their activation specificities, the strength of their activity in particular tissues or developmental stages appeared to determine whether activation actually occurred. Our findings provide the first evidence that plant‐derived enhancer/promoters can distantly interact/interfere with one another, which could pose potential problems for the tissue‐specific engineering of multiple traits using a single‐vector stacking approach. Therefore, our work highlights the importance of adopting enhancer‐blocking insulators in transformation vectors to minimize promoter–promoter interactions. The practical and fundamental significance of these findings will be discussed.     

A model for gain of function in superoxide dismutase

Studies have found that mutant, misfolded superoxide dismutase [Cu–Zn] (SOD1) can convert wild type SOD1 (wtSOD1) in a prion-like fashion, and that misfolded wtSOD1 can be propagated by release and uptake of protein aggregates. In developing a prion-like mechanism for this propagation of SOD1 misfolding we have previously shown how enervation of the SOD1 electrostatic loop (ESL), caused by the formation of transient non-obligate SOD1 oligomers, can lead to an experimentally observed gain of interaction (GOI) that results in the formation of SOD1 amyloid-like filaments. It has also been shown that freedom of ESL motion is essential to catalytic function. This work investigates the possibility that restricting ESL mobility might not only compromise superoxide catalytic activity but also serve to promote the peroxidase activity of SOD1, thus implicating the formation of SOD1 oligomers in both protein misfolding and in protein oxidation.     

A mouse model engineered to conditionally express the progesterone receptor‐B isoform

Using a Rosa26 gene targeting strategy in mouse embryonic stem cells, we have generated a new transgenic mouse (Pgr‐B ^LSL), which is designed to conditionally express the epitope‐tagged mouse progesterone receptor‐B (PGR‐B) isoform when crossed with a specific cre driver mouse. To functionally validate this transgenic mouse, we crossed the Pgr‐B ^LSL mouse with the MMTV‐CREA transgenic mouse to create the MMTV‐CREA/Pgr‐B ^LSL bigenic (termed PR‐B:OE to denote PGR‐B o vere xpressor). As expected, transgene‐derived PGR‐B protein was specifically targeted to the virgin mammary gland epithelium. At a functional level, the PR‐B:OE bigenic exhibited abnormal mammary morphogenesis—dilated epithelial ducts, precocious alveologenesis and lateral side‐branching, along with a prominent proliferative signature—that resulted in pregnant PR‐B:OE mice unable to exhibit mammary gland terminal differentiation at parturition. Because of this developmental failure, the PR‐B:OE mammary gland was incapable of producing milk resulting in early neonatal death of otherwise healthy litters. This first line of analysis demonstrates the utility of the Pgr‐B ^LSL mouse to examine the role of the PGR‐B isoform in different physiologic and pathophysiologic systems that are responsive to progesterone.     

Axin2‐mTurquoise2: A novel reporter mouse model for the detection of canonical Wnt signalling

The canonical Wnt signalling pathway has been implicated in organogenesis and self‐renewal of essentially all stem cell systems. In vivo reporter systems are crucial to assess the role of Wnt signalling in the biology and pathology of stem cell systems. We set out to develop a Turquoise (TQ) fluorescent protein based Wnt reporter. We used a CRISPR‐Cas9 approach to insert a TQ fluorescent protein encoding gene into the general Wnt target gene Axin2, thereby establishing a Wnt reporter mouse similar to previously generated Wnt reporter mice but with the mTurquoise2 gene instead of E. coli‐β‐galactosidase (LacZ). The use of mTurquoise2 is especially important in organ systems in which cells need to a be alive for further experimentation such as in vitro activation or transplantation studies. We here report successful generation of Axin2‐TQ mice and show that cells from these mice faithfully respond to Wnt signals. High Wnt signals were detected in the intestinal crypts, a classical Wnt signalling site in vivo, and by flow cytometry in the thymus. These mice are an improved tool to further elucidate the role of Wnt signalling in vivo.     

Novel behavioural characteristics of the superoxide dismutase 1 G93A (SOD1G93A) mouse model of amyotrophic lateral sclerosis include sex‐dependent phenotypes

Amyotrophic lateral sclerosis (ALS) involves the rapid degeneration of upper and lower motor neurons leading to weakening and paralysis of voluntary movements. Mutations in copper‐zinc superoxide dismutase 1 (SOD1) are a known genetic cause of ALS, and the SOD1 ^G93A mouse has been used extensively to investigate molecular mechanisms in ALS. In recent years, evidence suggests that ALS and frontotemporal dementia form a spectrum disorder ranging from motor to cognitive dysfunctions. Thus, we tested male and female SOD1 ^G93A mice for the first time before the onset of debilitating motor impairments in behavioural domains relevant to both ALS and frontotemporal dementia. SOD1 ^G93A males displayed reduced locomotion, exploration and increased anxiety‐like behaviours compared with control males. Intermediate‐term spatial memory was impaired in SOD1 ^G93A females, whereas long‐term spatial memory deficits as well as lower acoustic startle response, and prepulse inhibition were identified in SOD1 ^G93A mice of both sexes compared with respective controls. Interestingly, SOD1 ^G93A males exhibited an increased conditioned cue freezing response. Nosing behaviours were also elevated in both male and female SOD1 ^G93A when assessed in social paradigms. In conclusion, SOD1 ^G93A mice exhibit a variety of sex‐specific behavioural deficits beyond motor impairments supporting the notion of an ALS‐frontotemporal spectrum disorder. Thus, SOD1 ^G93A mice may represent a useful model to test the efficacy of therapeutic interventions on clinical symptoms in addition to declining motor abilities.     

Establishment of a mouse model for post‐inflammatory hyperpigmentation

Post‐inflammatory hyperpigmentation (PIH) is a common cutaneous condition that can cause a disfigured appearance. However, the pathophysiology of PIH remains poorly understood, at least in part, because an appropriate animal model for research has not been established. In order to analyze the pathomechanism of PIH, we successfully induced PIH in a hairless version of transgenic mice (hk14‐SCF Tg/HRM) that have a human‐type epidermis containing melanin by repeated hapten application of 2,4‐dinitrofluorobenzene. Histopathologic observation showed epidermal hyperplasia, predominant infiltrations of inflammatory cells, and melanin‐containing cells in the dermis just after elicitation of the atopic dermatitis‐like condition. At week 2, the findings were similar to the characteristics of PIH, that is, an increase of melanin without spongiosis or liquid degeneration in the epidermis and an increase in dermal melanophages. Dynamic analysis of melanin showed that the melanin in the dermis remained for a longer duration than in the epidermis. Furthermore, immunohistochemical staining revealed that the majority of cells containing melanin were positive for the anti‐CD68 antibody, but negative for the anti‐F4/80 antibody. These data suggest that novel treatments of PIH should be targeted against macrophages and should eventually lead to the development of new treatment modalities.     

A misfolded dimer of Cu/Zn‐superoxide dismutase leading to pathological oligomerization in amyotrophic lateral sclerosis

Misfolding of mutant Cu/Zn‐superoxide dismutase (SOD1) is a pathological hallmark in a familial form of amyotrophic lateral sclerosis. Pathogenic mutations have been proposed to monomerize SOD1 normally adopting a homodimeric configuration and then trigger abnormal oligomerization of SOD1 proteins. Despite this, a misfolded conformation of SOD1 leading to the oligomerization at physiological conditions still remains ambiguous. Here, we show that, around the body temperature (～37°C), mutant SOD1 maintains a dimeric configuration but lacks most of its secondary structures. Also, such an abnormal SOD1 dimer with significant structural disorder was prone to irreversibly forming the oligomers crosslinked via disulfide bonds. The disulfide‐crosslinked oligomers of SOD1 were detected in the spinal cords of the diseased mice expressing mutant SOD1. We hence propose an alternative pathway of mutant SOD1 misfolding that is responsible for oligomerization in the pathologies of the disease.     

Tissue‐specific and pathogen‐inducible expression of a fusion protein containing a Fusarium‐specific antibody and a fungal chitinase protects wheat against Fusarium pathogens and mycotoxins

Fusarium head blight (FHB) in wheat and other small grain cereals is a globally devastating disease caused by toxigenic Fusarium pathogens. Controlling FHB is a challenge because germplasm that is naturally resistant against these pathogens is inadequate. Current control measures rely on fungicides. Here, an antibody fusion comprised of the Fusarium spp.‐specific recombinant antibody gene CWP2 derived from chicken, and the endochitinase gene Ech42 from the biocontrol fungus Trichoderma atroviride was introduced into the elite wheat cultivar Zhengmai9023 by particle bombardment. Expression of this fusion gene was regulated by the lemma/palea‐specific promoter Lem2 derived from barley; its expression was confirmed as lemma/palea‐specific in transgenic wheat. Single‐floret inoculation of independent transgenic wheat lines of the T₃ to T₆ generations revealed significant resistance (type II) to fungal spreading, and natural infection assays in the field showed significant resistance (type I) to initial infection. Gas chromatography–mass spectrometry analysis revealed marked reduction of mycotoxins in the grains of the transgenic wheat lines. Progenies of crosses between the transgenic lines and the FHB‐susceptible cultivar Huamai13 also showed significantly enhanced FHB resistance. Quantitative real‐time PCR analysis revealed that the tissue‐specific expression of the antibody fusion was induced by salicylic acid drenching and induced to a greater extent by F. graminearum infection. Histochemical analysis showed substantial restriction of mycelial growth in the lemma tissues of the transgenic plants. Thus, the combined tissue‐specific and pathogen‐inducible expression of this Fusarium‐specific antibody fusion can effectively protect wheat against Fusarium pathogens and reduce mycotoxin content in grain.     

Generation of Elf5‐Cre knockin mouse strain for trophoblast‐specific gene manipulation

Placental development is a complex and highly controlled process during which trophoblast stem cells differentiate to various trophoblast subtypes. The early embryonic death of systemic gene knockout models hampers the investigation of these genes that might play important roles during placentation. A trophoblast specific Cre mouse model would be of great help for dissecting out the potential roles of these genes during placental development. For this purpose, we generate a transgenic mouse with the Cre recombinase inserted into the endogenous locus of Elf5 gene that is expressed specifically in placental trophoblast cells. To analyze the specificity and efficiency of Cre recombinase activity in Elf5‐Cre mice, we mated Elf5‐Cre mice with Rosa26^mT/mG reporter mice, and found that Elf5‐Cre transgene is expressed specifically in the trophoectoderm as early as embryonic day 4.5 (E4.5). By E12.5, the activity of Elf5‐Cre transgene was detected exclusively in all derivatives of trophoblast lineages, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. In addition, Elf5‐Cre transgene was also active during spermatogenesis, from spermatids to mature sperms, which is consistent with the endogenous Elf5 expression in testis. Collectively, our results provide a unique tool to delete specific genes selectively and efficiently in trophoblast lineage during placentation.     

Reduction of reperfusion‐induced ventricular fibrillation and infarct size via heme oxygenase‐1 overexpression in isolated mouse hearts

Heme oxygenase‐1 (HO‐1), also known as heat shock protein 32 (hsp‐32) is a stress‐induced cytoprotective protein. The present investigation evaluated the capacity of HO‐1 to reduce the incidence of reperfusion‐induced ventricular fibrillation (VF) and infarct size. HO‐1 transgenic (Tg) mice were generated using a rat HO‐1 genomic transgene. Isolated mouse hearts obtained from Tg and non‐transgenic (NTg) groups were exposed to 20 min. of global ischemia and 120 min. of reperfusion. Epicardial electrocardiogram was recorded to monitor the incidence of reperfusion‐induced VF and at the end of the reperfusion period, detection of HO‐1 by immunohistochemistry and measurement of infarct size using the tetrazolium chloride method were carried out. Results shown here provide additional support for cardioprotective effects of HO‐1 as demonstrated by the reduced infarct size. Moreover, overexpression of the HO‐1 efficiently reduced the incidence of ischemia/reperfusion induced VF in HO‐1 Tg mice.     

Tamoxifen‐inducible podocyte‐specific iCre recombinase transgenic mouse provides a simple approach for modulation of podocytes in vivo

We report the generation and initial characterization of a mouse line expressing tamoxifen‐inducible improved Cre (iCre) recombinase (iCre‐ER^T2) under the regulation of NPHS2 (podocin) gene promoter. The resulting transgenic mouse line was named podocin‐iCreER^T2 mice. The efficiency of iCre activity was confirmed by crossing podocin‐iCreER^T2 with the ROSA26 reporter mouse. By using the floxed ROSA reporter mice, we found that tamoxifen specifically induced recombination in the kidneys. In the absence of tamoxifen, recombination was undetectable in podocin‐iCreER^T2;ROSA26 mice. However, following intraperitoneal injection of tamoxifen, selective recombination was observed in the podocytes of adult animals. We further examined the efficiency of recombination by assessing various tamoxifen exposure regimens in adult mice. These results suggest that podocin‐iCre‐ER^T2 mouse provides an excellent genetic tool to examine the function of candidate genes in podocytes in a spatially and temporally‐restricted manner. genesis 48:446–451, 2010. © 2010 Wiley‐Liss, Inc.     

A mouse model with tamoxifen‐inducible thyrocyte‐specific cre recombinase activity

We have created a mouse model expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the thyroglobulin (Tg) gene promoter to be able to study the role of defined genetic modifications in the regulation of thyroid function. We chose the thyroglobulin promoter, as it is expressed specifically in the thyroid. In order to obtain reliable expression under the control of the Tg promoter, we used a P1 artificial chromosome (PAC) containing a large piece of the Tg promoter. A tamoxifen inducible CreER^T2 construct was selected to avoid the possible consequences of the gene deletion for the development of the thyroid gland, and to study the role of gene deletion in the adult thyroid. Transgenic lines (TgCreER^T2) carrying this construct were generated and analyzed by crossing the TgCreER^T2 mice with the ROSA26LacZ reporter strain. The activity and specificity of the Cre recombinase was tested by staining for β‐galactosidase activity and by immunohistochemistry using an anti‐Cre‐antibody. In the TgCreER^T2xROSA26LacZ reporter line, Cre‐mediated recombination occurred specifically in the thyrocytes only after tamoxifen administration, and no significant staining was observed in controls. The recombination efficiency was nearly complete, since almost all thyrocytes showed X‐gal staining. We could also induce the recombination in utero by giving tamoxifen to the pregnant female. In addition, mice expressing TgCreER^T2 had no obvious histological changes, hormonal alterations, or different response to growth stimuli as compared to controls. These results demonstrate that the TgCreER^T2 mouse line is a powerful tool to study temporally controlled deletion of floxed genes in the thyroid. genesis 52:333–340, 2014. © 2014 Wiley Periodicals, Inc.     

Immune responses in a mouse model of vitiligo with spontaneous epidermal de‐ and repigmentation

To generate a mouse model of spontaneous epidermal depigmentation, parental h3TA2 mice, expressing both a human‐derived, tyrosinase‐reactive T‐cell receptor on T cells and the matching HLA‐A2 transgene, were crossed to keratin 14‐promoter driven, stem cell factor transgenic (K14‐SCF) mice with intra‐epidermal melanocytes. In resulting Vitesse mice, spontaneous skin depigmentation precedes symmetrical and sharply demarcated patches of graying hair. Whereas the SCF transgene alone dictates a greater retinoic acid receptor‐related orphan receptor gamma (RORγt)⁺ T‐cell compartment, these cells displayed markedly increased IL‐17 expression within Vitesse mice. Similar to patient skin, regulatory T cells were less abundant compared with K14‐SCF mice, with the exception of gradually appearing patches of repigmenting skin. The subtle repigmentation observed likely reflects resilient melanocytes that coexist with skin‐infiltrating, melanocyte‐reactive T cells. Similar repigmenting lesions were found in a different TCR transgenic model of vitiligo developed on an SCF transgenic background, supporting a role for SCF in repigmentation.     

A transgenic red fluorescent protein‐expressing nude mouse for color‐coded imaging of the tumor microenvironment

The tumor microenvironment (TME) is critical for tumor growth and progression. We have previously developed color‐coded imaging of the TME using a green fluorescent protein (GFP) transgenic nude mouse as a host. However, most donor sources of cell types appropriate for study in the TME are from mice expressing GFP. Therefore, a nude mouse expressing red fluorescent protein (RFP) would be an appropriate host for transplantation of GFP‐expressing stromal cells as well as double‐labeled cancer cells expressing GFP in the nucleus and RFP in the cytoplasm, thereby creating a three‐color imaging model of the TME. The RFP nude mouse was obtained by crossing non‐transgenic nude mice with the transgenic C57/B6 mouse in which the β‐actin promoter drives RFP (DsRed2) expression in essentially all tissues. In crosses between nu/nu RFP male mice and nu/+ RFP female mice, the embryos fluoresced red. Approximately 50% of the offspring of these mice were RFP nude mice. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, duodenum, the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart, and major arteries and veins. The skinned skeleton highly expressed RFP. The bone marrow and spleen cells were also RFP positive. GFP‐expressing human cancer cell lines, including HCT‐116‐GFP colon cancer and MDA‐MB‐435‐GFP breast cancer were orthotopically transplanted to the transgenic RFP nude mice. These human tumors grew extensively in the transgenic RFP nude mouse. Dual‐color fluorescence imaging enabled visualization of human tumor–host interaction. The RFP nude mouse model should greatly expand our knowledge of the TME. J. Cell. Biochem. 106: 279–284, 2009. © 2008 Wiley‐Liss, Inc.     

A new variant of serum leucine aminopeptidase in the mouse: Its development and possible regulation

Serum leucine aminopeptidase (LAP) isozymes were compared in four strains of inbred mice during postnatal development, adult life, and pregnancy. In pregnancy, no changes in the maternal serum LAP pattern were observed, in contrast to human studies. One strain, DD/S, differs from the other three in serum LAP. Polymorphism in serum LAP has not been previously described in the mouse. Neonatal DD/S mice exhibit a single band of serum LAP upon starch gel electrophoresis; however, between 14 and 18 days of age, two distinct bands appear, which persist throughout adult life. In the strains C57BL/6J, BALB/cJ, and DBA/2J there is a single band of activity at all stages. Crosses and backcrosses between DD/S and C57BL/6J show that the double-band variant is inherited as an autosomal recessive. The variant is independent of both the supernatant malic enzyme (Mod-1) and the intestinal LAP (Lap-1) loci, which are known to be linked on chromosome 9. The serum LAP variant is linked to an intestinal alkaline phosphatase variant. The presence of a separate structural gene is suggested by the genetic independence of the serum LAP variant from Lap-1. Also, the two serum LAP bands of DD/S are not interconverted by treatment with neuraminidase, -mercaptoethanol, or heat or by mixing the sera of DD/S and C57BL/6J prior to electrophoresis. The level of serum LAP activity in DD/S is approximately twice that in C57BL/6J. While these observations imply two structurally distinct proteins, the absence of any trace of the second LAP band in the heterozygote strongly suggests that the LAP variant protein is not the result of a separate structural gene. Intestinal LAP in DD/S migrates with the same electrophoretic mobility as the serum LAP variant, implying that the variant might originate in the intestine and its appearance in the serum be modulated by some factor at an unlinked locus.This work was supported by National Institutes of Health Grant RR08117.     

A damped precipitation‐driven,bottom‐up model for deer mouse population abundance in the northwestern United States

Small‐mammal population densities can be regulated by bottom‐up (food availability) and top‐down (predation) forces. In 1993, an El Niño Southern Oscillation event was followed by a cluster of human hantavirus with pulmonary syndrome in the southwestern United States. An upward trophic cascade hypothesis was proposed as an explanation for the outbreak: Increased plant productivity as a consequence of El Niño precipitations led to an unusual increase in distribution and abundance of deer mice (Peromyscus maniculatus ; reservoir host of Sin Nombre virus). Could such drastic events occur in mesic habitats, where plant productivity in response to climate conditions is likely to be much less dramatic? In this work, we investigate to what extent deer mouse populations follow a precipitation‐driven, bottom‐up model in central and western Montana and discuss important conditions for such a model to be possible. We found positive correlations between deer mouse abundance and on‐the‐ground measured plant productivity with a several‐month lag in three of six study sites. This effect was weaker when deer mouse populations were more abundant, indicating density‐dependent effects. Dispersal resulting from territoriality may be important in attenuating local density increments in spite of high food availability. In addition, there is evidence that population abundance in the study area could respond to other abiotic factors. In particular, precipitation in the form of snow may reduce deer mice survival, thus compensating the benefits of improved plant productivity. Deer mouse populations in Montana study sites follow complex dynamics determined by multiple limiting factors, leading to a damped precipitation‐driven bottom‐up regulation. This prevents dramatic changes in rodent abundances after sudden increments of food availability, such as those observed in other regions.     

Seed‐specific overexpression of antioxidant genes in Arabidopsis enhances oxidative stress tolerance during germination and early seedling growth

Enzymatic and non‐enzymatic antioxidants play important roles in the tolerance of abiotic stress. To increase the resistance of seeds to oxidative stress, At2S3 promoter from Arabidopsis was used to achieve overexpression of the antioxidants in a seed‐specific manner. This promoter was shown to be capable of driving the target gene to have a high level of expression in seed‐related organs, including siliques, mature seeds, and early seedlings, thus making its molecular farming applications in plants possible. Subsequently, genes encoding Mn‐superoxide dismutase (MSD1), catalase (CAT1), and homogentisate phytyltransferase (HPT1, responsible for the first committed reaction in the tocopherol biosynthesis pathway) were overexpressed in Arabidopsis under the control of the At2S3 promoter. Double overexpressers co‐expressing two enzymes and triple overexpressers were produced by cross pollination. Mn‐SOD and total CAT activities, as well as γ‐tocopherol content, significantly increased in the corresponding overproduction lines. Moreover, single MSD1‐transgene, double, and triple overexpressers displayed remarkably enhanced oxidative stress tolerance compared to wild type during seed germination and early seedling growth. Interestingly, an increase in the total CAT activity was also observed in the single MSD1‐transgenic lines as a result of MSD1 overexpression. Together, the combined increase in Mn‐SOD and CAT activities in seeds plays an essential role in the improvement of antioxidant capacity at early developmental stage in Arabidopsis.