Tail Biopsies Research Articles

Adenosine A2A Receptor Signaling and Golf Assembly Show a Specific Requirement for the γ7 Subtype in the Striatum

The adenosine A(2A) receptor (A(2A)R) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α(olf) subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ(7) subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ(7) protein controls the hierarchial assembly of a specific G-protein α(olf)β(2)γ(7) heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A(2A)R activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ(7) subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A(2A)R G-protein α(olf)β(2)γ(7) interface as a possible therapeutic target for Parkinson disease.

Porcine Induced Pluripotent Stem Cells Produce Chimeric Offspring

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.

Assessing the efficacy of protein farnesyltransferase inhibitors in mouse models of progeria

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the accumulation of a farnesylated form of prelamin A (progerin). Previously, we showed that blocking protein farnesylation with a farnesyltransferase inhibitor (FTI) ameliorates the disease phenotypes in mouse model of HGPS (LmnaHG/+). However, the interpretation of the FTI treatment studies is open to question in light of recent studies showing that mice expressing a nonfarnesylated version of progerin (LmnanHG/+) develop progeria-like disease phenotypes. The fact that LmnanHG/+ mice manifest disease raised the possibility that the beneficial effects of an FTI in LmnaHG/+ mice were not due to the effects of the drug on the farnesylation of progerin, but may have been due to unanticipated secondary effects of the drug on other farnesylated proteins. To address this issue, we compared the ability of an FTI to improve progeria-like disease phenotypes in both LmnaHG/+ and LmnanHG/+ mice. In LmnaHG/+ mice, the FTI reduced disease phenotypes in a highly significant manner, but the drug had no effect in LmnanHG/+ mice. The failure of the FTI to ameliorate disease in LmnanHG/+ mice supports the idea that the beneficial effects of an FTI in LmnaHG/+ mice are due to the effect of drug on the farnesylation of progerin.

Non-invasive genotyping of transgenic mice: comparison of different commercial kits and required amounts

The usefulness of genetically modified mice is discussed critically. Nevertheless, their number has increased dramatically over the past years. A principle for the use these mice is the isolation of DNA to determine whether an individual carries the genetic modification or not. This "genotyping" is usually done by invasive tissue sampling, e.g. tail biopsies, which likely causes discomfort and pain to the animals and is therefore discussed in animal welfare regulations. Although non-invasive tissue sampling by stool samples was already described over 10 years ago, it is not commonly used so far. In the present study we therefore tested the practicality of this method by the use of three commercial kits for genotyping and compared the results to those attained using tail biopsies. Our data shows that DNA isolation from stool samples is practical, sensitive and effective. In addition, the possibility of repeated sampling represents a clear advantage to invasive genotyping. Therefore, this method represents a useful tool in the 3R concept, since replacement of invasive tissue sampling refines the use of transgenic mice.

Wound Healing in the Flight Membranes of Big Brown Bats

Biologists routinely punch the flight membranes of bats to collect tissue for molecular analyses, or to mark animals in the field, or both. The current standard is to biopsy the wing membrane (chiropatagium) because it is easy to access and is less vascularized, and thus bleeds less, than the tail membrane (uropatagium). Although flight membrane biopsies are assumed not to affect the bat's ability to fly or capture prey, almost nothing is known about wound healing times and the optimal punch size or location for tissue excision. We measured wound healing in the wing and tail membrane of 32 big brown bats (Eptesicus fuscus) biopsied with 2 circular punch tool sizes, and quantified the concentration of DNA extracted from the excised tissue. Our results show that tail wounds healed significantly faster than wing wounds for both 4-mm- and 8-mm-diameter biopsy wounds. We also were able to extract significantly more DNA from tail biopsies than from wing biopsies of the same size. The newly healed tissue remains unpigmented for considerable time after wound closure, and this allows identification of individuals for an extended period. We hypothesize that the increased vasculature in the uropatagium contributes to faster healing times compared to the chiropatagium. Examination of our data indicates that tissue biopsy for molecular analyses in bats should be taken from the tail membrane, although biopsies of the wing membrane are useful for marking associated with recapture programs because the wound and scar will persist longer.

An Inducible Cartilage Oligomeric Matrix Protein Mouse Model Recapitulates Human Pseudoachondroplasia Phenotype

Cartilage oligomeric matrix protein (COMP) is a pentameric extracellular protein expressed in cartilage and other musculoskeletal tissues. Mutations in the COMP gene cause pseudoachondroplasia (PSACH), a severe dwarfing condition that has a growth plate chondrocyte pathology. PSACH is characterized by intracellular retention of COMP and other extracellular matrix (ECM) proteins, which form an ordered matrix within large rough endoplasmic reticulum cisternae. This accumulation is cytotoxic and causes premature chondrocyte cell death, thereby depleting chondrocytes needed for normal long bone growth. Research to define the underlying molecular mechanisms of PSACH has been hampered by the lack of a suitable model system. In this study, we achieved robust expression of human mutant (MT) or wild-type (WT) COMP in mice by using a tetracycline-inducible promoter. Normal growth plate distribution of ECM proteins was observed in 1-month-old WT-COMP and C57BL\6 control mice. In contrast, the structure of the MT-COMP growth plate recapitulated the findings of human PSACH growth plate morphology, including (1) retention of ECM proteins, (2) intracellular matrix formation in the rER cisternae, and (3) increased chondrocyte apoptosis. Therefore, we have generated the first mouse model to show extensive intracellular retention of ECM proteins recapitulating the human PSACH disease process at the cellular level.

MMP-13 Plays a Role in Keratinocyte Migration, Angiogenesis, and Contraction in Mouse Skin Wound Healing

Matrix metalloproteinases (MMPs) have been implicated in wound healing. To analyze the roles of MMP-9 and MMP-13 in wound healing, we generated full-thickness cutaneous wounds in MMP-9 knockout (KO), MMP-13 KO, MMP-9/13 double KO, and wild-type mice. Macroscopic wound closure was delayed in all of the KO mice, as compared with wild-type mice. The rate of re-epithelialization was significantly delayed in MMP-9 KO and MMP-13 KO mice and remarkably delayed in MMP-9/13 double KO mice, as compared with wild-type mice. Both MMP-9 and MMP-13 were expressed by the leading edges of epidermal cells in wild-type mice, and the migration of keratinocytes was suppressed by treatment with an MMP inhibitor or transfection of small interfering RNAs for MMP-9 or MMP-13, as compared with controls. The vascular density in wound granulation was significantly lower in both MMP-13 KO and MMP-9/13 double KO mice than in wild-type mice. Degradation of connective tissue growth factor in wound tissue was transiently prevented in MMP-13 KO mice. Morphometric analyses demonstrated a reduction in both wound contraction and myofibroblast formation in both MMP-13 KO and MMP-9/13 double KO mice. Proliferation and transforming growth factor-beta1-induced myofibroblast differentiation of dermal fibroblasts from MMP-13 KO mice were decreased, as compared with wild-type dermal fibroblasts. These data suggest that MMP-13 plays a role in keratinocyte migration, angiogenesis, and contraction in wound healing, while MMP-9 functions in keratinocyte migration.

The Effects of Tail Biopsy for Genotyping on Behavioral Responses to Nociceptive Stimuli

Removal of a small segment of tail at weaning is a common method used to obtain tissue for the isolation of genomic DNA to identify genetically modified mice. When genetically manipulated mice are used for pain research, this practice could result in confounding changes to the animals' responses to noxious stimuli. In this study, we sought to systematically investigate whether tail biopsy representative of that used in standard genotyping methods affects behavioral responses to a battery of tests of nociception. Wild-type littermate C57BL/6J and 129S6 female and male mice received either tail biopsies or control procedural handling at Day 21 after birth and were then tested at 6–9 weeks for mechanical and thermal sensitivity. C57BL/6J mice were also tested in the formalin model of inflammatory pain. In all tests performed (von Frey, Hargreaves, modified Randall Selitto, and formalin), C57BL/6J tail-biopsied animals' behavioral responses were not significantly different from control animals. In 129S6 animals, tail biopsy did not have a significant effect on behavioral responses in either sex to the von Frey and the modified Randall-Selitto tests of mechanical sensitivity. Interestingly, however, both sexes exhibited small but significant differences between tail biopsied and control responses to a radiant heat stimulus. These results indicate that tail biopsy for genotyping purposes has no effect on nocifensive behavioral responses of C57BL/6J mice, and in 129S6 mice, causes only a minor alteration in response to a radiant heat stimulus while other nocifensive behavioral responses are unchanged. The small effect seen is modality- and strain-specific.

Rapid and reliable determination of transgene zygosity in mice by multiplex ligation-dependent probe amplification

The ability to rapidly and unequivocally distinguish heterozygous from homozygous transgenic mice is an integral part of any breeding strategy. Here we describe a quick and simple protocol for determining the zygosity of transgenic mice at multiple loci in a single reaction. This involved the development of a multiplex ligation-dependent probe amplification (MLPA) probe mix to simultaneously measure common transgenic alleles such as Cre recombinase (Cre), neomycin (Neo), beta-galactosidase (LacZ) and enhanced green fluorescent protein (eGFP), as well as loci specific to the X and Y chromosome to allow sexing. Each reaction required as little as 100 ng of genomic DNA isolated from a tail biopsy using a simple procedure. Normalization against autosomal control loci resulted in 100% call accuracy, with no ambiguous results. This probe mix can be easily implemented in any laboratory with access to a PCR machine and a DNA sequencer, and can be rapidly adapted to genotype any additional loci of interest.

Mice Lacking the Extracellular Matrix Protein WARP Develop Normally but Have Compromised Peripheral Nerve Structure and Function

WARP is a recently identified extracellular matrix molecule with restricted expression in permanent cartilages and a distinct subset of basement membranes in peripheral nerves, muscle, and the central nervous system vasculature. WARP interacts with perlecan, and we also demonstrate here that WARP binds type VI collagen, suggesting a function in bridging connective tissue structures. To understand the in vivo function of WARP, we generated a WARP-deficient mouse strain. WARP-null mice were healthy, viable, and fertile with no overt abnormalities. Motor function and behavioral testing demonstrated that WARP-null mice exhibited a significantly delayed response to acute painful stimulus and impaired fine motor coordination, although general motor function was not affected, suggesting compromised peripheral nerve function. Immunostaining of WARP-interacting ligands demonstrated that the collagen VI microfibrillar matrix was severely reduced and mislocalized in peripheral nerves of WARP-null mice. Further ultrastructural analysis revealed reduced fibrillar collagen deposition within the peripheral nerve extracellular matrix and abnormal partial fusing of adjacent Schwann cell basement membranes, suggesting an important function for WARP in stabilizing the association of the collagenous interstitial matrix with the Schwann cell basement membrane. In contrast, other WARP-deficient tissues such as articular cartilage, intervertebral discs, and skeletal muscle showed no detectable abnormalities, and basement membranes formed normally. Our data demonstrate that although WARP is not essential for basement membrane formation or musculoskeletal development, it has critical roles in the structure and function of peripheral nerves.

Prolactin–Growth Factor Crosstalk Reduces Mammary Estrogen Responsiveness Despite Elevated ERα Expression

Most breast cancers that occur in women express estrogen receptor alpha (ERalpha). However, a large subset of these cancers either does not initially respond to anti-estrogen therapy or develops resistance to such treatment modalities. One postulated mechanism of this failure is signaling cross talk between hormones and local growth factors. To examine these complex interactions in vivo, we assessed the effects of estrogen on transforming growth factor alpha (TGFalpha)- and prolactin (PRL)-induced mammary tumorigenesis in transgenic mice. Both PRL and estrogen reduced the latency of TGFalpha-induced oncogenesis, resulting in tumors that were variably ERalpha-positive, but were progesterone receptor-negative. However, despite elevated ERalpha levels in NRL-PRL/TGFalpha glands, tumor latency was not reduced with increasing estrogen levels, nor increased after ovariectomy. Furthermore, PRL and TGFalpha in combination blocked the mitogenic effects of estrogen, dramatically reduced progesterone receptor levels, and diminished ERalpha down-regulation in response to circulating estrogen levels, in contrast to the other genotypes. Notably, however, ductal morphology remained responsive to estrogen, indicating that TGFalpha and PRL in combination can inhibit some, but not all, estrogenic signals. Both in vitro and in vivo, PRL and TGFalpha cooperatively enhanced Akt phosphorylation, which is associated with endocrine resistance in human disease. These findings provide insight into the interactions of PRL with growth factors during mammary oncogenesis and suggest combinatorial approaches that may result in improved therapeutic efficacy.

The V-ATPase B1-subunit promoter drives expression of Cre recombinase in intercalated cells of the kidney

The collecting duct of the kidney is composed of two morphologically and physiologically distinct cell types, principal and intercalated cells. To better understand intercalated cell function we generated a transgenic mouse expressing Cre recombinase under the control of a cell type- specific promoter. We used 7 kb of the ATP6V1B1 5' untranslated region (B1 promoter), a gene found in the intercalated cells of the kidney and the male reproductive tract. We first crossed these B1-Cre transgenic mice with the ROSA26-loxP-stop-loxP-yellow fluorescent protein reporter mice to assess the specificity of Cre expression. Immunohistochemistry and confocal fluorescence microscopy showed that Cre is selectively active in all intercalated cells (type A, type B, and non-A/B cells) within the collecting duct and most cells of the connecting segment. About half of the principal cells of the connecting segment also expressed Cre, a pattern also seen in B1-driven enhanced green fluorescent protein transgenic mice. Cre was found to be active in the male reproductive tract and at a low level in limited non-ATP6V1B1 expressing tissues. The B1-Cre transgenic mice are healthy, breed normally, produce regular sized litters, and transmit the transgene in Mendelian fashion. This new cell-specific Cre expressing mouse should prove useful for the study of intercalated cell physiology and development.

Trichinella zimbabwensis in wild Nile crocodiles ( Crocodylus niloticus) of South Africa

Recent discovery of Trichinella zimbabwensis in crocodiles from Zimbabwe, Lake Cahora Basa, Mozambique, and from lake Abaja, Ethiopia, prompted strict control measures to curb the possible spread of the infection to humans and also to prevent its introduction to other countries, which were considered free of this pathogen. In 2006, the Chief Directorate Veterinary Services of Mpumalanga Province of South Africa launched a survey to investigate the status of wild and commercial breeding crocodiles in the province. To evaluate if T. zimbabwensis was circulating in the environments where crocodiles are living in South Africa, 9 fish, 36 reptiles (including 27 Nile crocodiles) and 4 mammals have been investigated to detect Trichinella sp. larvae in their muscles. In January 2008, a Nile crocodile from Komatipoort, sampled by means of a tail biopsy, tested positive for Trichinella larvae. In June–July 2008, Trichinella sp. larvae were also detected in four other Nile crocodiles from the Olifants River Gorge. The prevalence of Trichinella infection in the investigated wild Nile crocodiles from South Africa is 38.5%. The larvae were identified as belonging to T. zimbabwensis by multiplex-PCR. These are the first reports of T. zimbabwensis in South Africa and suggest that the distribution area of this parasite species is wider than that believed in the past.

Removal of the N-terminal Extension of Cardiac Troponin I as a Functional Compensation for Impaired Myocardial β-Adrenergic Signaling

Although beta-adrenergic stimuli are essential for myocardial contractility, beta-blockers have a proven beneficial effect on the treatment of heart failure, but the mechanism is not fully understood. The stimulatory G protein alpha-subunit (Gsalpha) couples the beta-adrenoreceptor to adenylyl cyclase and the intracellular cAMP response. In a mouse model of conditional Gsalpha deficiency in the cardiac muscle (Gsalpha-DF), we demonstrated heart failure phenotypes accompanied by increases in the level of a truncated cardiac troponin I (cTnI-ND) from restricted removal of the cTnI-specific N-terminal extension. To investigate the functional significance of the increase of cTnI-ND in Gsalpha-DF cardiac muscle, we generated double transgenic mice to overexpress cTnI-ND in Gsalpha-DF hearts. The overexpression of cTnI-ND in Gsalpha-DF failing hearts increased relaxation velocity and left ventricular end diastolic volume to produce higher left ventricle maximum pressure and stroke volume. Supporting the hypothesis that up-regulation of cTnI-ND is a compensatory rather than a destructive myocardial response to impaired beta-adrenergic signaling, the aberrant expression of beta-myosin heavy chain in adult Gsalpha-DF but not control mouse hearts was reversed by cTnI overexpression. These data indicate that the up-regulation of cTnI-ND may partially compensate for the cardiac inefficiency in impaired beta-adrenergic signaling.

Role of H2-calponin in Regulating Macrophage Motility and Phagocytosis

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.

Targeted Ablation of Glucose-dependent Insulinotropic Polypeptide-producing Cells in Transgenic Mice Reduces Obesity and Insulin Resistance Induced by a High Fat Diet

The K cell is a specific sub-type of enteroendocrine cell located in the proximal small intestine that produces glucose-dependent insulinotropic polypeptide (GIP), xenin, and potentially other unknown hormones. Because GIP promotes weight gain and insulin resistance, reducing hormone release from K cells could lead to weight loss and increased insulin sensitivity. However, the consequences of coordinately reducing circulating levels of all K cell-derived hormones are unknown. To reduce the number of functioning K cells, regulatory elements from the rat GIP promoter/gene were used to express an attenuated diphtheria toxin A chain in transgenic mice. K cell number, GIP transcripts, and plasma GIP levels were profoundly reduced in the GIP/DT transgenic mice. Other enteroendocrine cell types were not ablated. Food intake, body weight, and blood glucose levels in response to insulin or intraperitoneal glucose were similar in control and GIP/DT mice fed standard chow. In contrast to single or double incretin receptor knock-out mice, the incretin response was absent in GIP/DT animals suggesting K cells produce GIP plus an additional incretin hormone. Following high fat feeding for 21-35 weeks, the incretin response was partially restored in GIP/DT mice. Transgenic versus wild-type mice demonstrated significantly reduced body weight (25%), plasma leptin levels (77%), and daily food intake (16%) plus enhanced energy expenditure (10%) and insulin sensitivity. Regardless of diet, long term glucose homeostasis was not grossly perturbed in the transgenic animals. In conclusion, studies using GIP/DT mice demonstrate an important role for K cells in the regulation of body weight and insulin sensitivity.

Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens

Many diverse human diseases are associated with protein aggregation in ordered fibrillar structures called amyloid. Amyloid formation may mediate aberrant protein interactions that culminate in neurodegeneration in Alzheimer, Huntington, and Parkinson diseases and in prion encephalopathies. Studies of protein aggregation in the brain are hampered by limitations in imaging techniques and often require invasive methods that can only be performed postmortem. Here we describe transgenic mice in which aggregation-prone proteins that cause Huntington and Parkinson disease are expressed in the ocular lens. Expression of a mutant huntingtin fragment or alpha-synuclein in the lens leads to protein aggregation and cataract formation, which can be monitored in real time by noninvasive, highly sensitive optical techniques. Expression of a mutant huntingtin fragment in mice lacking the major lens chaperone, alphaB-crystallin, markedly accelerated the onset and severity of aggregation, demonstrating that the endogenous chaperone activity of alphaB-crystallin suppresses aggregation in vivo. These novel mouse models will facilitate the characterization of protein aggregation in vivo and are being used in efficient and economical screens for chemical and genetic modifiers of disease-relevant protein aggregation.

Protein Kinase Cϵ Regulates γ-Aminobutyrate Type A Receptor Sensitivity to Ethanol and Benzodiazepines through Phosphorylation of γ2 Subunits

Ethanol enhances gamma-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABA(A) receptors, especially at low concentrations achieved during social drinking. We postulated that the epsilon isoform of protein kinase C (PKCepsilon) regulates the ethanol sensitivity of GABA(A) receptors, as mice lacking PKCepsilon show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCepsilon mutant to selectively inhibit the catalytic activity of PKCepsilon. We used this mutant and PKCepsilon(-/-) mice to determine that PKCepsilon phosphorylates gamma2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at alpha1beta2gamma2 receptors, which is the most abundant GABA(A) receptor subtype in the brain. Our findings indicate that PKCepsilon phosphorylation of gamma2 regulates the response of GABA(A) receptors to specific allosteric modulators, and, in particular, PKCepsilon inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.

A Novel Genotyping Strategy Based on Allele-specific Inverse PCR for Rapid and Reliable Identification of Conditional FADD Knockout Mice

The apoptotic adapter protein FADD has been shown to play diverse roles in cell survival and proliferation. FADD knockout embryos died of heart defects, rendering Cre/loxP-mediated conditional FADD knockout mice a unique tool for investigating FADD-dependent nonapoptotic mechanism. Previously, these genetically engineered mice were identified by time-consuming Southern blot or controversial real-time PCR. In this article, we report a novel genotyping strategy based on allele-specific inverse PCR (ASI-PCR) for rapid and reliable identification of conditional FADD knockout mice. In this strategy, the knockout nature of FADD was simply identified by screening the absence of the wild type FADD-specific ASI-PCR product. Using this method, we accurately identified CD4-Cre-mediated T cell specific FADD knockout mice. The whole process can be accomplished in any normal biological laboratory within 12 h using genomic DNA from tail biopsy. The proposed ASI-PCR-based approach is simple, rapid, sensitive, reproducible, and especially suitable for genotyping small amount of spatiotemporally restricted biopsies and large animal population. We believe that the strategy described in this article may be of general utility in genotyping other conditional gene knockout mice.

The Mast Cell-restricted Tryptase mMCP-6 Has a Critical Immunoprotective Role in Bacterial Infections

Although it has been shown that mast cell-deficient mice have diminished innate immune responses against bacteria, the most important immunoprotective factors secreted from activated mast cells have not been identified. Mouse mast cell protease 6 is a tetramer-forming tryptase. This serine protease is abundant in the secretory granules and is exocytosed upon bacterial challenge. Here we have described the generation of a mast cell protease-6-null mouse. Our discovery that mice lacking this neutral protease cannot efficiently clear Klebsiella pneumoniae from their peritoneal cavities reveals an essential role for this serine protease, and presumably its human ortholog, in innate immunity.