Tail Biopsies Research Articles

Comparing Genotyping Accuracy Using Buccal Swabs versus Tail Biopsies by PCR in B6;C3-Tg(Prnp-SNCA*A53T)83Vle and B6;C3-Tg(Prnp-SNCA*A53T)83Vle Sncatm1Mjff Mice.

Genotyping is a common and necessary procedure performed on genetically modified animals to distinguish carriers from noncarriers of the variants of interest. Established methods involve collection of tissues such as tips of tails or notches of ears. Noninvasive methods have been described but not widely adopted for reasons including inertia to change, needs to adjust PCR protocols, and the lack of validation; noninvasive genotyping methods are a refinement on animal welfare, but questions remain regarding how they compare with invasive methods in terms of genotyping accuracy rate and reproducibility. To gain answers to these questions, we compared the detection accuracy of the transgene and determination of zygosity in B6;C3-Tg(Prnp-SNCA*A53T)83Vle and B6;C3-Tg(Prnp-SNCA*A53T)83Vle Snca tm1Mjff neonatal mice between tail biopsies and buccal swabs. Moreover, we weighed and observed mice following genotyping to see if any clinical differences can be discerned. Weight data did not support statistically significant differences in mice undergoing different genotyping procedures and control. No statistically significant difference was found between using buccal swabs or tail biopsies for genotyping with PCR or quantitative PCR. None of the pups swabbed was rejected by the dam. Our findings indicate that buccal swabbing is a more humane and feasible alternative to tail biopsies for high-throughput genotyping.

Genetic Absence of Integrin alpha2b Improves Survival of Tissue Factor Pathway Inhibitor Null Mice but Results in Hydrocephalus Formation

Introduction: Genetic deletion of Tissue Factor Pathway Inhibitor (TFPI) exon 4, encoding its Kunitz 1 (K1) domain, results in complete intrauterine lethality (PMID 9242522). TFPI K1 null mice (Tfpi-/-) are born live if Tissue Factor expression is reduced or in the complete absence of Protease Activated Receptor-4 (Par4), and these exhibit a normal life span without overt signs of thrombosis (PMID 15598816, 25954015). Based on these data, it has been postulated that modulation of thrombin-dependent platelet activation by TFPI is essential for survival. Platelet activation results in a number of downstream events. Of these, platelet aggregation via the integrin receptor αIIbβ3 is considered to have a key role in hemostasis and could participate in thrombotic pathology in the absence of TFPI. Binding of αIIbβ3 to its ligands also mediates critical interactions of platelets with endothelial cells, leukocytes and other cell types. In this work, we have investigated whether modulation of platelet activity via genetic absence of integrin receptor αIIbβ3 confers protection and allows generation of adult Tfpi-/- mice. Methods: Tfpi+/- αIIb-/- mice were generated by breeding Tfpi+/- and integrin αIIb-/- mice and identified by PCR-based genotyping of tissues obtained by tail biopsies. Tfpi+/- αIIb-/- intercrosses served as the experimental cross. Pups were genotyped at the time of wean, around 4 weeks of age. In some experiments, surgeries were performed to analyze pregnancies at 18.5 days post coitum (dpc). Embryos and placentae were observed under the dissecting microscope and any phenotypic abnormalities were noted. Presence of heart beats and limb movements were used to identify live embryos. Embryos and placentae were fixed in zinc-formalin and embedded in paraffin for sectioning and histological analysis. T1 weighted Magnetic Resonance Images were acquired on a 9.4T scanner to measure cerebral ventricle sizes of Tfpi-/- αIIb-/- and littermate control mice. Ventricular regions of interest (ROI) were drawn on each image slice from which total ventricular volume was computed. These mice were later perfused with 4% paraformaldehyde for collection of organs and histological analysis. Results: We analyzed 122 pups from intercrosses of Tfpi+/- αIIb-/- mice and observed a genetic distribution 39 Tfpi+/+, 77 Tfpi+/- and 6 Tfpi-/- (25% or 31 Tfpi-/- were expected, 5% observed, 95% CI 1.8 to 10.4%). Thus, genetic absence of αIIb results in incomplete rescue of Tfpi-/- mice (P< 0.000002, Χ2 GOF) with only ~20% surviving past embryonic development to 4 weeks of age. These data contrast with 40% Tfpi-/- offspring surviving in the absence of Par4 (Tfpi+/- Par4-/- intercrosses: 23 Tfpi+/+, 39 Tfpi+/- and 8 Tfpi-/-; PMID 25954015). Thus, the absence of αIIb is much less effective than the absence of Par4 in allowing early survival of TFPI null mice (P < 1.9E-09; Χ2 test of independence). We compared survival of Tfpi-/- αIIb-/- offspring close to term of pregnancy (18.5 dpc) and at 4 weeks of age. TFPI null embryos were found at reduced frequency at 18.5 dpc (12 Tfpi+/+, 21 Tfpi+/- and 5 Tfpi-/-; 25% Tfpi-/- expected, 13% observed, 95% CI 4.4 to 28.1%), but even fewer survived the trauma of birth (P<0.0004, Χ2 test of independence). Of the 6 Tfpi-/- αIIb-/- pups found at 4 weeks of age, 3 died by 9 weeks of age. Dome shaped heads indicative of hydrocephalus or histological evidence of hydrocephalus was noted in the pups that died. Surviving mice were observed for 7 months to 1 year of age and imaged with MRI. Comparison of ventricular volumes between Tfpi-/- mice and Tfpi+/- controls demonstrated a higher volume in Tfpi-/- mice (39.3 ± 18.3 versus 5.3 ± 2.2 mm3; P=0.08). Both Tfpi-/- and Tfpi+/- mice were αIIb-/- in this experiment. Thus, αIIb in not involved in hydrocephalus formation. Hydrocephalus formation in Tfpi-/- mice was confirmed through serial histological sections of the brain (Figure 1). Conclusions: Our data demonstrates that genetic absence of αIIb improves survival of TFPI null mice, but to a much lesser extent than the genetic absence of Par4. Thus, the critical role of Par4 in the demise of TFPI null mice is unlikely to be primarily through excessive platelet aggregation. We further show that TFPI null pups exhibit varying degrees of hydrocephalus formation. While the mechanism of hydrocephalus formation in the absence of TFPI remains unclear, our results demonstrate a critical role of TFPI in the brain. Disclosures No relevant conflicts of interest to declare.

Deficiency of Protease Activated Receptor 3 in the Mother Prevents Placenta-Mediated Death of Endothelial Protein C Receptor Null Mice, but Uncovers an Obligate Requirement of EPCR in the Embryo and Neonate

Introduction: Genetically engineered deletion of ProcR, the gene encoding EPCR, results in early embryonic death, secondary to developmental and functional abnormalities in the placenta (PMID 12218060). ProcR-null embryonic death is prevented by either genetically reducing TF expression or maintaining extraembryonic ProcR expression in placental trophoblast cells (PMID 15956290). These results indicate that placental abnormalities observed in ProcR-null mice are the result of unregulated thrombin generation. Thrombin activates platelets through cleavage of Protease Activated Receptors, Par3 and Par4, present on the surface of murine platelets. The objective of this work was to examine whether decreased responsiveness of maternal platelets to thrombin due to the loss of Par3 would be sufficient to overcome placental abnormality and allow the generation of live ProcR-null mice. Methods: Par3-/- ProcR+/- mice were generated by breeding Par3-/- and ProcR+/- mice and identified by PCR-based genotyping of tissues obtained by tail biopsies. In experimental crosses, ProcR+/- males were used to sire pregnancies of Par3-/- ProcR+/- females. Reverse genetic crosses, pregnancies of ProcR+/- females sired by Par3-/- ProcR+/- males, served as control. Surgeries were conducted to analyze embryonic survival and placental phenotypes at various stages of gestation. Presence of heart beats and limb movements were used to identify live embryos. We also employed a previously published Meox2Cre-Lox system which selectively ablates ProcR in the embryo while maintaining extraembryonic expression (PMID 15956290). Meox2Cretg/-ProcR-/- mice were generated through this system. Results: As previously reported, ProcR-/- mice from ProcR+/- intercrosses die on or before 10.5 days post coitum (dpc) (Table 1, Row 1: 25% or 10 ProcR-/- expected, 0.0% observed, 95% CI: 0.0 to 8.6%). ProcR-/- embryos were present in expected Mendelian proportions in pregnancies of Par3-/- ProcR+/- mothers at 11.5 dpc (Table 1, Row 2: 25% or 8 ProcR-/- expected, 26.7% observed, 95% CI: 12.9 to 45.9%) and at 18.5 dpc (Table 1, Row 3: 25% or 9 ProcR-/- expected, 25% observed, 95% CI: 12.1 to 42.2%). Thus, genetic absence of Par3 in the mother completely rescues ProcR-/- mice from intrauterine death. In contrast, in preliminary studies no live ProcR-/- embryos have so far been observed in pregnancies of ProcR+/- females sired by Par3-/- ProcR+/- males at 11.5 dpc (4 ProcR+/+, 5 ProcR+/-, 0 ProcR-/-; 7 abortions; 25% ProcR-/- expected, 0.0% observed, CI: 0.0 to 33.6%), suggesting that paternal loss of Par3 does not confer the same protection. We further noted that ProcR-/- embryos and placentae were significantly smaller than their ProcR+/- and ProcR+/+ littermate controls (Figure 1 and 2). To our surprise, we found that Par3+/- ProcR-/- progeny of Par3-/- ProcR+/- females were present at reduced frequency as neonates (Table 1, Row 4: 25% or 19 ProcR-/- expected, 13.2% observed, 95% CI: 6.5 to 22.9%) and most did not survive till wean (Table 1, Row 5: 25% or 11 ProcR-/-expected, 2.2% observed, 95% CI: 0.06 to 11.8%). These observations contrast with the published fate of Meox2Cretg/- ProcR-/- mice generated by maintaining ProcR expression on placental trophoblast cells (PMID 15956290) and replicated by us for comparison. As previously reported, we observed 27 Meox2Cre-/- ProcRlox/- and 23 Meox2Cretg/- ProcR-/- at wean (~4 weeks of age) from pregnancies of ProcRlox/lox females sired by Meox2Cretg/- ProcRlox/- males (P= 0.85, χ2 GOF test; 50% expected, 46.0% observed, 95% CI: 31.8 to 60.7%). Conclusions: Our data demonstrates that the placenta is highly sensitive to local thrombin concentrations and maternal loss of Par3 is sufficient to overcome the obligate requirement of EPCR in the placenta. Second, we show that in stark contrast to ProcR-deficient mice generated through Cre-Lox technology for embryo-specific knockout, germ line deleted ProcR mice rescued through the maternal absence of Par3 die during the neonatal period. These data uncover a critical role of ProcR in postembryonic survival. Disclosures No relevant conflicts of interest to declare.

Surveillance of phlebotomine fauna and Didelphis marsupialis (Didelphimorphia: Didelphidae) infection in an area highly endemic for visceral leishmaniasis in Colombia.

The study of the interaction between the parasite, the vector and the mammalian hosts, including man, allows to understand the behavior of the leishmaniases. To determine the presence of Lutzomyia species and to detect the Leishmania infection in Didelphis marsupialis in an endemic area for visceral leishmaniasis. Phlebotomine fauna and individuals of D. marsupialis were collected with CDC and Tomahawk™ traps, respectively. The species of Lutzomyia were identified using the Young and Duncan key (1994). Ear and tail biopsies and blood samples from D. marsupialis were taken to identify the Leishmania species by amplifying a fragment of the gene associated with the 70 kD heat shock protein. Seven Lutzomyia species were identified: Lu. evansi, Lu. gomezi, Lu. panamensis, Lu. dubitans, Lu. cayennensis cayennensis, Lu. rangeliana and Lu. trinidadensis. The first three species have epidemiological importance in Colombia because of their implications in the transmission of the Leishmania parasite. Sixty-five tissue samples from 19 D. marsupialis individuals were negative for Leishmania spp. The presence of the Lutzomyia species that have been identified as vectors for Leishmania inside and around houses in the village of El Bledo, in El Carmen de Bolívar represents a risk of infection. Furthermore, the presence of Lu. panamensis is reported for first time in El Carmen de Bolívar in Colombia. Although the lack of detection of Leishmania spp. in D. marsupialis samples may suggest that D. marsupialis does not play an important role in the transmission cycle of Leishmania in this region, it is necessary to carry out further longitudinal studies to confirm this hypothesis.

A Noninvasive Ocular (Tear) Sampling Method for Genetic Ascertainment of Transgenic Mice and Research Ethics Innovation.

Animal models, animal welfare and research ethics are both facilitators and gatekeepers for Big Data generation in genomics and multi-omics R&D. Safeguarding animal welfare is also a research ethics issue that can benefit from technical innovations in biosample collection in particular. Animal welfare draws from the guiding principles of 3R, namely, "Replacement" (methods avoiding the use of animals in research), "Reduction" (methods using fewer animals or derive more information from the same number of animals), and "Refinement" (methods removing or minimizing pain or distress). We report here that noninvasive ocular (tear) sampling for genetic ascertainment of transgenic mice can serve as an innovative ethical safeguard for animal welfare, and as a veritable alternative to the surgical tail biopsies, ear puncture, or blood sampling from the weanling transgenic mice. We compared ocular versus tail biopsy sampling in regard to ascertainment, by genotyping, of apolipoprotein E-deficient (ApoE-/-) transgenic weanling mice (n = 60) by one-round polymerase chain reaction analysis. We found that ocular sampling compares to the results obtained by tail sampling with the obvious benefit of being noninvasive and improving the 3R, especially for the Refinement principle of animal welfare. To place the importance of this new biosample collection approach into further context, transgenic mice research and animal models are at the epicenter of Big Data translation to health innovation. We suggest that ocular sampling is considered and evaluated further in transgenic mice models, not to mention warrant exploration for applications in other types of animal models that require noninvasive biosample collection.

ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination.

Deubiquitination of NLRP3 has been suggested to contribute to inflammasome activation, but the roles and molecular mechanisms are still unclear. We here demonstrate that ABRO1, a subunit of the BRISC deubiquitinase complex, is necessary for optimal NLRP3-ASC complex formation, ASC oligomerization, caspase-1 activation, and IL-1β and IL-18 production upon treatment with NLRP3 ligands after the priming step, indicating that efficient NLRP3 activation requires ABRO1. Moreover, we report that ABRO1 deficiency results in a remarkable attenuation in the syndrome severity of NLRP3-associated inflammatory diseases, including MSU- and Alum-induced peritonitis and LPS-induced sepsis in mice. Mechanistic studies reveal that LPS priming induces ABRO1 binding to NLRP3 in an S194 phosphorylation-dependent manner, subsequently recruiting the BRISC to remove K63-linked ubiquitin chains of NLRP3 upon stimulation with activators. Furthermore, deficiency of BRCC3, the catalytically active component of BRISC, displays similar phenotypes to ABRO1 knockout mice. Our findings reveal an ABRO1-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome and provide novel potential targets for treating NLRP3-associated inflammatory diseases.

RNAi-Mediated Loss of Function of Xenopus Immune Genes by Transgenesis.

Generation of transgenic frogs through the stable integration of foreign DNA into the genome is well established in Xenopus This protocol describes the combination of transgenesis with stable RNA interference as an efficient reverse genetic approach to study gene function in Xenopus Initially developed in the fish medaka and later adapted to Xenopus, this transgenic method uses the I-SceI meganuclease, a "rare-cutter" endonuclease with an 18 bp recognition sequence. In this protocol, transgenic X. laevis with knocked down expression of a specific gene are generated using a double promoter expression cassette. This cassette, which is flanked by I-SceI recognition sites, contains the shRNA of choice under the control of the human U6 promoter and a green fluorescent protein (GFP) reporter gene under the control of the human EF-1α promoter. Prior to microinjection the plasmid is linearized by digestion with I-SceI and the entire reaction is then microinjected into one-cell stage eggs. The highly stringent recognition sequence of I-SceI is thought to maintain the linearized plasmid in a nonconcatamerized state, which promotes random integration of the plasmid transgene in the genome. The injected embryos are reared until larval stage 56 and then screened for GFP expression by fluorescence microscopy and assessed for effective knockdown by quantitative RT-PCR using a tail biopsy. Typically, the I-SceI meganuclease transgenesis technique results in 35%-50% transgenesis efficiency, a high survival rate (>35%) and bright nonmosaic GFP expression. A key advantage of this technique is that the high efficiency and nonmosaic transgene expression permit the direct use of F0 animals.

Structural and functional analysis of CRISP3 protein in relation to diseases of exocrine pancreas

Introduction: Human cysteine rich secretory protein 3 (CRISP3), also known as specific granule protein (SGP28) is a small 28kDa protein. CRISP-3 is widely expressed in salivary glands, pancreas (particularly in pancreatic disease), lachrymal glands, prostate, myeloid precursors, human epididymis, ovary, thymus and colon. Human CRISP3 share similarity with snake venoms as well as Glioma Pathogenesis Related protein GLIPR1. Here we are reporting the extent of alignment of human CRISP3 protein with pancreatic enzymes and snake venom proteins and presence of GLIPR1 and CRISP3 genes containing conserved GHYTQVVW motifs in clinical samples of pancreatitis and pancreatic cancer. Material and Methods: Protein sequences with accession numbers for Human CRISP3, Snake Venom and enzymes present in pancreatic juices i.e. Trypsinogen, Cathepsin, Lipase, Amylase and Elastase were retrieved from swissprot, protein database. These sequences were aligned in silico for demonstrating the presence of 16 conserved cysteine residues and other motifs i.e. GENL and GHYTQVVW. The multiple sequence alignment (MSA) was performed. Presence of CRISP-3 conserved motifs in clinical samples of pancreatitis and pancreatic cancer was evaluated by PCR. DNA extracted from a small piece taken from inflamed pancreatic tail biopsy with confirmed diagnosis for pancreatitis and an excised pancreatic cancer tissue was subjected to PCR to conform presence of CRISP3 and GLIPR1 genes. Results: Our results show conservation of 9 Cysteine residues out of 16 Cysteine residues in majority of pancreatic enzymes and notable conservation of GENL and GHYTQVVW motifs across snake venom proteins. PCR yielded desired amplicon of expected size with the primers spanning conserved residues and core consensus motifs specifically designed to amplify CRISP3 gene in clinical samples of pancreatitis and pancreatic cancer. We also detected presence of GLIPR1 gene sequence in the DNA extracted from clinical samples of pancreatitis and pancreatic cancer thus confirming similarity between the pathogenesis related protein and CRISP3. Conclusion: Notable conservation of cysteine residues across pancreatic enzymes, snake venom and Human CRISP3 opens avenues of further research to evaluate the likely clinical potential of the conserved residues as putative biomarkers in exocrine pancreatic diseases. To the best of our knowledge this is the first report where primers designed around conserved GHYTQVVW motifs to amplify CRISP3 gene and its structural similarity with GLIPR1 gene sequence in clinical samples of pancreatitis and pancreatic cancer has been validated.

Abstract 5126: Does genetic background in glioma influence the sensitivity of tumor cells to a PARP inhibitor in the presence of different DNA damaging agents

Abstract Background: Upregulation of cellular signaling pathways for proliferation is one of the characteristic features of high-grade glioblastomas (GBM), the most common and lethal type of adult brain tumors. Common oncogenic alterations in GBM include a characteristic gain-of-function of growth-promoting EGFR gene, a functionally relevant activation of p21-RAS pathway or a loss-of-expression of growth-attenuating tumor suppressor PTEN gene. Here we tested the role of genetic backgrounds (RAS pathway upregulation and PTEN nullness) on the sensitivity of ABT888 in the presence of either temozolomide (TMZ) or carboplatin (C). Methods: We examined the anti-tumor effects of ABT888 using PTEN null U87MG cell line and a transgenic mouse model (12V-Ha-Ras transgenic mice) of human glioma expressing V12 RAS (Can. Res. 2001). LacZ, GFAP and PCNA-positive primary astrocytes (derivative astrocytes, passage 100) were used for the study. The effect of the drugs were tested by (1) soft agar colony growth (2) 3D- On-Top assay (3) cell cycle analyses (4) vascular mimicry (real time) and (5) Western blots in a dose and time dependent manner. Results: Expression of the transgene in 12V-Ha-Ras mice (tail biopsy of mice from which derivative glioma cells cultures were established) and in the derivative glioma cells (established from the transgene-expressing mice) at different passages were confirmed by genotyping. Testing for astrocytic marker GFAP by ICC (>95% GFAP positive at passage 10 and onwards). Both U87MG glioma cells and astrocytoma cells from 12V-Ha-Ras mice were positive for GFAP and PCNA. Our results show that (1) the combination of ABT888 plus TMZ is additive in reducing soft agar colony growth in both U87MG and V12RAS astrocytes (2) ABT888 sensitized both U87MG and V12RAS astrocytes to TMZ in 3D On-Top assay, (3) in both U87MG and V12RAS astrocytes the largest amount of G0 was observed following the combination of ABT888 plus C at 72 hours (4) ABT888 plus C as well as ABT888 plus TMZ blocked vascular mimicry in both U87MG and V12RAS astrocytes and (5) at 72 hours PTEN null U87MG cells exhibited an increase in pro-apoptotic cl-PARP and a decrease in anti-apoptotic MCL-1 following both combination treatments, an effect not observed in V12RAS astrocytes. At 48 hours the TMZ induced increase in PAR is blocked by ABT888, an effect more pronounced in U87MG cells. Conclusion: The data demonstrate that ABT888 sensitizes the effect of TMZ in PTEN null glioma cells. The functional link between tumor suppressor, PTEN and the sensitization of PARP inhibitor is being currently worked out utilizing glioma cells expressing PTEN to specifically address the role of PTEN nullness in the context of ABT888 in combination with TMZ, the results of which will be presented at the meeting. Citation Format: Jennifer H. Carlson, Brian Leyland-Jones, David Martin, Pradip De, Nandini Dey. Does genetic background in glioma influence the sensitivity of tumor cells to a PARP inhibitor in the presence of different DNA damaging agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5126. doi:10.1158/1538-7445.AM2014-5126

Biological characteristics of cleft palate relevant gene thyroid transcription factor-2 transgenic mice

The aim of this study is to establish a transgenic mouse model for cleft palate relevant gene thyroid transcription factor-2 (TTF-2), which can be used to study palatal shelf development when the expression pattern and regular activation of TTF-2 is altered. The C57BL/6J mouse TTF-2 gene was cloned through polymerase chain reaction (PCR) from the mouse genomic DNA. The TTF-2 gene was inserted into the expression vector pBROAD3-mcs to construct the recombinant expression vector pBROAD3-TTF-2. This expression vector was then microinjected into the male pronuclei of the fertilized mouse ovum. Thus, the TTF-2 transgenic mice model was established. The genotype of the transgenic mice was identified by PCR and Southern blot analysis. Immunohistochemistry identified the consistent expression of TTF-2 gene during its palatal shelf development. TTF-2 genes were microinjected into 982 fertilized ova. A total of 580 two-cell-stage embryos cultured and transplanted into the oviducts of 48 pseudopregnant female mice. Overall, 68 embryos were obtained for analysis. The genotype of the mice was determined through PCR and Southern blot analysis using genomic DNA extracted from tail biopsies of the transgenic fetus. A total of 13 TTF-2 transgenic mice were detected. The expression of TTF-2 gene during the palatal shelf development of the transgenic mice was consistently detected by immunohistochemistry. The recombinant expression vector pBROAD3-TTF-2 was integrated into mouse genome through microinjection. The transgenic mouse in the palatal shelf that consistently expressed TTF-2 was successfully established and displayed a cleft palate phenotype.

Sensory Neuron Development in Mouse Coccygeal Vertebrae and Its Relationship to Tail Biopsies for Genotyping

A common method of genotyping mice is via tissue obtained from tail biopsies. However, there is no available information on the temporal development of sensory neurons in the tail and how their presence or absence might affect the age for performing tail biopsies. The goals of this study were to determine if afferent sensory neurons, and in particular nociceptive neurons, are present in the coccygeal vertebrae at or near the time of birth and if not, when they first can be visualized on or in those vertebrae. Using toluidine blue neuronal staining, transmission electron microscopy, and calcitonin-related gene peptide immunostaining, we found proximal to distal maturation of coccygeal nerve growth in the C57BL/6J mouse. Single nerve bundles were first seen on postpartum day (PPD) 0. On PPD 3 presumptive nociceptive sensory nerve fibers were seen entering the vertebral perichondrium. Neural development continued through the last time point (PPD 7) but at no time were neural fibers seen entering the body of the vertebrae. The effect of age on the development of pain perception in the neonatal mouse is discussed.

Farnesyltransferase Haplodeficiency Reduces Neuropathology and Rescues Cognitive Function in a Mouse Model of Alzheimer Disease

Isoprenoids and prenylated proteins have been implicated in the pathophysiology of Alzheimer disease (AD), including amyloid-β precursor protein metabolism, Tau phosphorylation, synaptic plasticity, and neuroinflammation. However, little is known about the relative importance of the two protein prenyltransferases, farnesyltransferase (FT) and geranylgeranyltransferase-1 (GGT), in the pathogenesis of AD. In this study, we defined the impact of deleting one copy of FT or GGT on the development of amyloid-β (Aβ)-associated neuropathology and learning/memory impairments in APPPS1 double transgenic mice, a well established model of AD. Heterozygous deletion of FT reduced Aβ deposition and neuroinflammation and rescued spatial learning and memory function in APPPS1 mice. Heterozygous deletion of GGT reduced the levels of Aβ and neuroinflammation but had no impact on learning and memory. These results document that farnesylation and geranylgeranylation play differential roles in AD pathogenesis and suggest that specific inhibition of protein farnesylation could be a potential strategy for effectively treating AD.

A Method for High-Throughput PCR-Based Genotyping of Larval Zebrafish Tail Biopsies

Here we describe a method for high-throughput genotyping of live larval zebrafish as early as 72 h post-fertilization (hpf). Importantly, this technique allows rapid and cost-effective PCR-based genotyping from very small fin biopsies, which regenerate as the embryo develops, thereby allowing researchers to select embryos with desired genotypes to be raised to adulthood.

VIPoma in a 37-year-old man

In September, 2011, a 37-year-old man with a past history of pulmonary tuberculosis came to our hospital with persistent watery diarrhoea, weight loss, and hypokalaemia of 2 years’ duration. The diarrhoea did not stop with fasting, and serum potassium had remained at 2∙5 mmol/L despite multiple cycles of intravenous potassium replacement. Physical examination and routine blood investigations were normal, as were both upper and lower gastrointestinal tract endoscopies. An abdominal CT showed a 5∙9 × 3∙4 cm lesion in the pancreatic tail and a solitary hepatic metastasis, confi rmed by MRI (fi gure). However, in view of the patient’s past history of tuberculosis, the medical team waited for mycobacterial culture results before making a diagnosis of probable stage IV pancreatic adeno carcinoma. Meanwhile, an endocrine referral was made to address the hypokalaemia. The patient’s signs and symptoms made the clinical team consider the extremely rare diagnosis of a vasoactive intestinal peptide endocrine tumour— VIPoma, also known as Verner-Morrison syndrome. We requested serum chromogranin A and 24-h urinary 5-hydoxyindoleacetic acid, which were both negative. The news of Steve Jobs’s death from a neuroendocrine tumour, which occurred around this time, prompted the clinical team to reconsider this diagnosis despite initial negative results. Endoscopic ultrasound-guided pancreatic tail biopsy was therefore done next, and immunohistochemistry of the tumour tissue stained positive for cytokeratin AE1/3, synaptophysin, and chromo granin. Blood samples were taken for vasoactive intestinal peptide ELISA assay, before a trial of octreotide was initiated. His diarrhoea resolved within 3 days, accompanied by the resolution of hypokalaemia. The plasma vasoactive intestinal peptide was 175 pmol/L (normal 3 cm in diameter. About 75% occur in the pancreatic tail, and 60–80% are already metastasised at diagnosis. Being slow growing, VIPomas are generally compatible with long survival of 5–10 years. Most adult patients are diagnosed between 30–50 years of age. High volume secretory diarrhoea (>700 mL/day) that persists with fasting is virtually universal. Hypo kalaemia occurs in 70–100% of patients. Investigations include serum vasoactive intestinal peptide, pancreatic polypeptide, and chromogranin A. Functional PET imaging such as 68-Ga DOTA fusion PET/CT scans have sensitivity of about 97%, compared with about 80% in CT scans, and 85% in MRIs. This higher accuracy facilitates much earlier detection of metastases. When used therapeutically, peptide receptor radionuclide therapy was reported in one case report to reduce widespread metastatic disease down to a single liver metastasis at 1-year follow-up. A limitation of peptide receptor radionuclide therapy is the potential lack of somatostatin receptor expression by the tumour. In such cases, sunitinib and everolimus (both in phase III trials) are among the most promising alternatives to traditional hepatic-directed therapies including resection, ablation, hepatic artery embolisation, and liver transplantation. Surgery remains the defi nitive treatment for VIPoma. Octreotide is useful in suppressing VIP secretion, and interferon-alpha and glucocorticoids are sometimes used in refractory cases to improve symptoms of hormonal secretion.

Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin

BackgroundDimeric human erythropoietin (dHuEPO) peptides are reported to exhibit significantly higher biological activity than the monomeric form of recombinant EPO. The objective of this study was to produce transgenic (tg) mice expressing dHuEPO and to investigate the characteristics of these mice.MethodsA dHuEPO-expressing vector under the control of the goat beta-casein promoter, which produced a dimer of human EPO molecules linked by a 2-amino acid peptide linker (Asp-Ile), was constructed and injected into 1-cell fertilized embryos by microinjection. Mice were screened using genomic DNA samples obtained from tail biopsies. Blood samples were obtained by heart puncture using heparinized tubes, and hematologic parameters were assessed. Using the microarray analysis tool, we analyzed differences in gene expression in the spleens of tg and control mice.ResultsA high rate of spontaneous abortion or death of the offspring was observed in the recipients of dHuEPO embryos. We obtained 3 founder lines (#4, #11, and #47) of tg mice expressing the dHuEPO gene. However, only one founder line showed stable germline integration and transmission, subsequently establishing the only transgenic line (#11). We obtained 2 F1 mice and 3 F2 mice from line #11. The dHuEPO protein could not be obtained because of repeated spontaneous abortions in the tg mice. Tg mice exhibited symptoms such as short lifespan and abnormal blood composition. The red blood cell count, white blood cell count, and hematocrit levels in the tg mice were remarkably higher than those in the control mice. The spleens of the tg mice (F1 and F2 females) were 11- and -21-fold larger than those of the control mice. Microarray analysis revealed 2,672 spleen-derived candidate genes; more genes were downregulated than upregulated (849/764). Reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) were used for validating the results of the microarray analysis of mRNA expression.ConclusionsIn conclusion, dHuEPO tg mice caused excessive erythrocytosis that led to abnormal blood composition, short lifespan, and abnormal splenomegaly. Further, we identified 2,672 genes associated with splenomegaly by microarray analysis. These results could be useful in the development of dHuEPO-producing tg animals.

Influence of Nitrate and Nitrite on Thyroid Hormone Responsive and Stress-Associated Gene Expression in Cultured Rana catesbeiana Tadpole Tail Fin Tissue

Nitrate and nitrite are common aqueous pollutants that are known to disrupt the thyroid axis. In amphibians, thyroid hormone (TH)-dependent metamorphosis is affected, although whether the effect is acceleration or deceleration of this developmental process varies from study to study. One mechanism of action of these nitrogenous compounds is through alteration of TH synthesis. However, direct target tissue effects on TH signaling are hypothesized. The present study uses the recently developed cultured tail fin biopsy (C-fin) assay to study possible direct tissue effects of nitrate and nitrite. Tail biopsies obtained from premetamorphic Rana catesbeiana tadpoles were exposed to 5 and 50 mg/L nitrate (NO3–N) and 0.5 and 5 mg/L nitrite (NO2–N) in the absence and presence of 10 nM T3. Thyroid hormone receptor β (TRβ) and Rana larval keratin type I (RLKI), both of which are TH-responsive gene transcripts, were measured using quantitative real time polymerase chain reaction. To assess cellular stress which could affect TH signaling and metamorphosis, heat shock protein 30, and catalase (CAT) transcript levels were also measured. We found that nitrate and nitrite did not significantly change the level of any of the four transcripts tested. However, nitrate exposure significantly increased the heteroscedasticity in response of TRβ and RLKI transcripts to T3. Alteration in population variation in such a way could contribute to the previously observed alterations of metamorphosis in frog tadpoles, but may not represent a major mechanism of action.

P4-03-05: Development of an Inducible Estrogen Receptor Co-Activator PELP1 Mammary Tumor Model.

Abstract Despite treatment advances, breast cancer remains the second most lethal malignant disease for women worldwide. Given the importance of estrogen receptor (ER) and hormone-dependent nature of breast cancer, pharmacologic agents were developed to either modulate ER functions or reduce circulating estrogens levels. Although targeted endocrine therapies significantly reduce mortality in patients with estrogen sensitive (ER+) tumors, both de novo and acquired resistance limits efficacy. A critical need for identifying more precise diagnostic/prognostic biomarkers and novel therapeutic targets prompted deeper investigation into ER-coregulatory protein function and regulation. ER-coactivator PELP1, mediates both nuclear and extra-nuclear estrogen signaling and crosstalk with growth factors. PELP1 is deregulated in hormone-driven cancers, associates with undifferentiated invasive breast adenocarcinomas and an independent prognostic biomarker in assessing clinical outcome of luminal-like breast cancer patients. Collectively, several studies suggest PELP1 is an ERα coregulator with tumorigenic potential. However, the in vivo significance of PELP1 deregulation during initiation and progression of breast cancer is unknown. To determine the role of PELP1 overexpression in mammary tumorigenesis, we generated an inducible transgenic murine model. Transgene construct (pTetOPELP1) consists of a full-length human PELP1 cDNA linked to luciferase gene reporter through an internal ribosomal entry site (IRES). PELP1 transgene was purified and microinjected into mouse zygotes to generate pTetOPELP1 mice. Founder mice were identified by Southern blot analysis, of genomic DNA extracted from tail biopsies, for transgene integration through germline transmission. pTetOPELP1 mice were breed with mammary gland-specific rtTA mice (MMTVrtTA) to produce MMTVrtTA-TetOPELP1 bitransgenic mice. Potential founder mice were identified by polymerase chain reaction and breed to establish two independent transgenic lines. Transgene expression was induced in adult female bitransgenic animals with 200mg/mL of doxycycline administered in drinking water. Concurrent expression and activity of the luciferase gene reporter was detected specifically in the mammary gland by in vivo bioluminescence imaging, luciferase assay and RT-PCR. Mammary epithelial-specific expression of PELP1 was validated by immunohistochemistry and Western blot analysis. PELP1-mediated morphological and histological changes were analyzed by examining carmine-stained whole mounts and H&E-stained mammary glands sections. Our early findings with MMTVrtTA-TetOPELP1 bitransgenic mice (n=30) early preneoplastic changes and hyperplasia were evident as early as 12 weeks and the formation of mammary tumors by 8 months of age following PELP1 induction by doxycycline treatment. By utilizing the tetracycline-regulatory system, we created a novel, inducible and mammary gland-specific PELP1-expressing transgenic model for future in vivo studies into molecular mechanisms of PELP1-mediated mammary tumorigenesis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-03-05.

Mutant HFE H63D Protein Is Associated with Prolonged Endoplasmic Reticulum Stress and Increased Neuronal Vulnerability

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability.

Basement Membrane Deposition of Nidogen 1 but Not Nidogen 2 Requires the Nidogen Binding Module of the Laminin γ1 Chain

The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (γ1III4) of the laminin γ1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in γ1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin γ3 chain, which contains a γ1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin γ1 and γ3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins.

Use of quantitative polymerase chain reaction analysis to compare quantity and stability of isolated murine DNA

Deoxyribonucleic acid (DNA) can be extracted from different tissue sources for genotyping of mice. Methods of collecting tissue vary with respect to their perceived invasiveness, and in some cases, tissue is collected multiple times in order to verify a genotype. The authors' goal was to refine and optimize tissue collection methods with quantitative measures for subsequent genotyping. To do this, the authors used real-time quantitative polymerase chain reaction (qPCR) analysis to quantify DNA extracted from fecal pellets, hair, buccal swab samples, ear punch samples and tail biopsy samples and then compared the quantities of DNA obtained by using either previously published protocols or commercial kits to extract DNA. They found that 2-mm tail biopsy samples yielded significantly more DNA than did the other sources and that commercial extraction kits generally yielded more DNA than did published protocols. The authors also assessed the stability of DNA extracted from the various tissue sources by repeating the qPCR analysis after the samples had been frozen and stored for 44 months. Although the quantities of DNA in the stored samples had decreased, all the samples could still be used for PCR-based genotyping. The authors' work supports the collection of a single minimal biopsy sample of 2 mm of tail or ear tissue, above other sources, for highest yield of DNA for genotyping. With appropriate storage, DNA remains usable for PCR-based genotyping for years without a need for repeated animal sampling.