Gene Targeting In ES Cells Research Articles

Bleeding Phenotype Observed in Murine Defect of VWF-Collagen 4 Interactions

VWF binding to type 4 collagen has recently been shown to occur via the VWF A1 domain, but the clinical implications of this interaction remain to be elucidated. In humans, the R1399H sequence variant (2% of the Caucasian population is heterozygous for this variant) abrogates VWF binding to collagen 4, while select other VWF A1 domain variants decrease VWF binding to collagen 4 to varying degrees. We generated a murine model of 1399H VWF to investigate the in vitro and in vivo effects of this variant more thoroughly.Mice expressing the murine R1399H mutation were generated via gene targeting in ES cells, generation of germline-competent ES cell chimeras, and backcrossing the resultant mutant mice onto a C57Bl6 background. VWF antigen (VWF:Ag) and VWF collagen binding with murine type 4 collagen (VWF:CB4) were measured by ELISA (n≥5 for each group) on wild-type p.R1399 mice (WT), heterozygous p.R1399H mice (R1399H) and homozygous p.H1399 mice (1399HH) and compared to mice lacking VWF entirely (VWF -/-). Tail bleeding time was performed by clipping a 3 mm segment of tail, immersing in warm saline, and monitoring bleeding for a maximum of 10 minutes (n≥4 for each group). Blood loss was also measured during the tail bleeding time assay for each mouse. VWF-dependent platelet adhesion to collagen 4 was measured under flow using the Venaflux system. Channels were coated with murine type 4 collagen overnight. Blood was drawn from the inferior vena cava, anticoagulated with PPACK and heparin, and platelets labeled with mepacrine. Whole blood samples were run over the collagen 4-coated channel at a shear rate of 1111/sec (n≥3 for each group). Images at 180 seconds following the start of flow over the chip were analyzed using ImageJ.Breeding of heterozygous and homozygous 1399H mice was observed to follow normal Mendelian ratios, indicating no deleterious effects of the 1399H variant on viability. No spontaneous bleeding was observed for any of the offspring. VWF expression as measured by VWF:Ag was similar for WT, R1399H, and 1399HH mice, but VWF:CB4/VWF:Ag ratios were decreased for R1399H mice, to an average of 55% of WT and 1399HH mice, to an average of 15% of WT. Blood loss during tail bleeding was increased compared to WT for both R1399H and 1399HH mice by approximately 1.8 fold and 2.8 fold respectively. Bleeding time also increased, with an average bleeding time of 92 seconds in the WT mice, 177 seconds in the R1399H mice, and 235 seconds in the 1399HH mice. Previous studies have demonstrated that platelet adhesion to collagen 4 under flow is dependent on VWF, with VWF -/- mice demonstrating absence of platelet adhesion. Platelet adhesion to collagen 4 was diminished in both heterozygous and homozygous 1399H mice, with maximum platelet binding intensity 61% of WT for the R1399H mice and 28% of WT for the 1388HH mice. These figures closely approximate the static collagen 4 binding as seen by comparable VWF:CB4/VWF:Ag ratios and platelet adhesion under flow for the R1399H heterozygous and 1399HH homozygous mice.These results show that a decrease in VWF's ability to bind collagen 4 under static conditions corresponds to a decrease in binding under flow conditions, at least at the measured shear rate, and that this defect also translates to increased bleeding time and blood loss in a tail clip model. This study supports the potential for a bleeding phenotype in patients with aberrant VWF-collagen 4 binding. Since 2% of the US population is heterozygous for the p.R1399H sequence variation, defective VWF interaction with collagen 4 may be a relatively common cause of clinical bleeding and can be modeled in engineered mice. DisclosuresFlood:CSL Behring: Consultancy; Baxter: Consultancy. Montgomery:Baxter: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy; Octapharma: Consultancy; Grifols: Consultancy; Biogen: Consultancy.

A mouse model for human deafness DFNB22 reveals that hearing impairment is due to a loss of inner hair cell stimulation

The gene causative for the human nonsyndromic recessive form of deafness DFNB22 encodes otoancorin, a 120-kDa inner ear-specific protein that is expressed on the surface of the spiral limbus in the cochlea. Gene targeting in ES cells was used to create an EGFP knock-in, otoancorin KO (Otoa(EGFP/EGFP)) mouse. In the Otoa(EGFP/EGFP) mouse, the tectorial membrane (TM), a ribbon-like strip of ECM that is normally anchored by one edge to the spiral limbus and lies over the organ of Corti, retains its general form, and remains in close proximity to the organ of Corti, but is detached from the limbal surface. Measurements of cochlear microphonic potentials, distortion product otoacoustic emissions, and basilar membrane motion indicate that the TM remains functionally attached to the electromotile, sensorimotor outer hair cells of the organ of Corti, and that the amplification and frequency tuning of the basilar membrane responses to sounds are almost normal. The compound action potential masker tuning curves, a measure of the tuning of the sensory inner hair cells, are also sharply tuned, but the thresholds of the compound action potentials, a measure of inner hair cell sensitivity, are significantly elevated. These results indicate that the hearing loss in patients with Otoa mutations is caused by a defect in inner hair cell stimulation, and reveal the limbal attachment of the TM plays a critical role in this process.

The Sox17‐mCherry fusion mouse line allows visualization of endoderm and vascular endothelial development

Sox17 is a HMG-box transcription factor that has been shown to play important roles in both cardio-vascular development and endoderm formation. To analyze these processes in greater detail, we have generated a Sox17-mCherry fusion (SCF) protein by gene targeting in ES cells. SCF reporter mice are homozygous viable and faithfully reflect the endogenous Sox17 protein localization. We report that SCF positive cells constitute a subpopulation in the visceral endoderm before gastrulation and time-lapse imaging reveals that SCF monitors the nascent definitive endoderm during epithelialization. After gastrulation, SCF marks the mid- and hindgut endoderm and vascular endothelial network, which can be imaged during establishment in allantois explant cultures. The SCF reporter is downregulated in the endoderm epithelium and upregulated in endothelial cells of the intestine, lung, and pancreas during organogenesis. In summary, the generation of the Sox17-mCherry reporter mouse line allows direct visualization of endoderm and vascular development in culture and the mouse embryo.

Design and Generation of Gene-Targeting Vectors.

This unit provides an overview of the major types of mutant alleles that can be generated by gene targeting in ES cells. It presents the growing public resources of premade gene targeting vectors, modified ES cells, and mutant mice. General guidelines for the design of targeting vectors are followed by protocols for the construction of vectors to generate knockout (KO), conditional KO, and subtle mutant alleles. Curr. Protoc. Mouse Biol. 1:199-211. © 2011 by John Wiley & Sons, Inc.

Angiotensin-converting Enzyme Overexpression in Mouse Myelomonocytic Cells Augments Resistance to Listeria and Methicillin-resistant Staphylococcus aureus

Gene targeting in ES cells was used to substitute control of angiotensin converting enzyme (ACE) expression from the endogenous promoter to the mouse c-fms promoter. The result is an animal model called ACE 10/10 in which ACE is overexpressed by monocytes, macrophages, and other myelomonocytic lineage cells. To study the immune response of these mice to bacterial infection, we challenged them with Listeria monocytogenes or methicillin-resistant Staphylococcus aureus (MRSA). ACE 10/10 mice have a significantly enhanced immune response to both bacteria in vivo and in vitro. For example, 5 days after Listeria infection, the spleen and liver of ACE 10/10 mice had 8.0- and 5.2-fold less bacteria than wild type mice (WT). In a model of MRSA skin infection, ACE 10/10 mice had 50-fold less bacteria than WT mice. Histologic examination showed a prominent infiltrate of ACE-positive mononuclear cells in the skin lesions from ACE 10/10. Increased bacterial resistance in ACE 10/10 is directly due to overexpression of ACE, as it is eliminated by an ACE inhibitor. Critical to increased immunity in ACE 10/10 is the overexpression of iNOS and reactive nitrogen intermediates, as inhibition of iNOS by the inhibitor 1400W eliminated all in vitro and in vivo differences in innate bacterial resistance between ACE 10/10 and WT mice. Increased resistance to MRSA was transferable by bone marrow transplantation. The overexpression of ACE and iNOS by myelomonocytic cells substantially boosts innate immunity and may represent a new means to address serious bacterial infections.

Inflammatory and age-related pathologies in mice with ectopic expression of human PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) is a sensor for DNA strand breaks and some unusual DNA structures and catalyzes poly(ADP-ribosyl)ation of nuclear proteins with NAD + serving as substrate. PARP-1 is involved in the regulation of genomic integrity, transcription, inflammation, and cell death. Due to its versatile role, PARP-1 is discussed both as a longevity factor and as an aging-promoting factor. Recently, we generated a mouse model with ectopic integration of full-length hPARP-1 [Mangerich, A., Scherthan, H., Diefenbach, J., Kloz, U., van der Hoeven, F., Beneke, S. and Bürkle, A., 2009. A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1. Transgenic Res. 18, 261–279]. Here, we show that hPARP-1 mice exhibit impaired survival rates accompanied by reduced hair growth and premature development of several inflammation and age-associated pathologies, such as adiposity, kyphosis, nephropathy, dermatitis, pneumonitis, cardiomyopathy, hepatitis, and anemia. Moreover, mutant male mice showed impaired glucose tolerance, yet without developing manifest diabetes. Overall tumor burden was comparable in wild-type and hPARP-1 mice, but tumor spectrum was shifted in mutant mice, showing lower incidence of sarcomas, but increased incidence of carcinomas. Furthermore, DNA repair was delayed in splenocytes of hPARP-1 mice, and gene expression of pro-inflammatory cytokines was dysregulated. Our results suggest that in hPARP-1 mice impaired DNA repair, accompanied by a continuous low-level increase in pro-inflammatory stimuli, causes development of chronic diseases leading to impaired survival.

PCP effector gene Inturned is an important regulator of cilia formation and embryonic development in mammals

The PCP effector gene Inturned regulates planar cell polarity (PCP) and wing hair formation in Drosophila wings. In order to understand the roles for Inturned in mammalian embryonic development, we generated a null mutant allele for the mouse homologue of Inturned (Intu) via gene-targeting in ES cells. Mouse Intu null mutants are homozygous lethal at midgestation, exhibiting multiple defects including neural tube closure defects, abnormal dorsal/ventral patterning of the central nervous system and abnormal anterior–posterior patterning of the limbs resulting in severe polydactyly (7–9 digits each limb). The developmental processes affected by the Intu mutation are under the control of Hh signaling through Gli-family transcription factors. We found that in Intu mutant embryos the expression of Gli1 and Ptch1, two direct transcriptional targets of Hh signaling, is down-regulated, and the proteolytic processing of Gli3 is compromised. We further demonstrate that Intu plays significant roles in the formation of primary cilia both during embryonic development and in cultured fibroblasts. Finally, a cytoplasmic GFP-Intu fusion protein efficiently rescues the ciliogenic defects in Intu mutant cells. In conclusion, we show that PCP effector gene Intu is an important regulator of cilia formation, Hh signal transduction, and embryonic development in mammals.

A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1

Here we report an approach to generate a knock-in mouse model using an 'ends-out' gene replacement vector to substitute the murine Parp-1 (mParp-1) coding sequence (32 kb) with its human orthologous sequence (46 kb). Unexpectedly, examination of mutant ES cell clones and mice revealed that site-specific homologous recombination was mimicked in three independently generated ES cell clones by bidirectional extension of the vector homology arms using the endogenous mParp-1-flanking sequences as templates. This was followed by adjacent integration of the targeting vector, thus leaving the endogenous mParp-1 locus functional. A related phenomenon termed 'ectopic gene targeting' has so far only been described for 'ends-in' integration-type vectors in non-ES cell gene targeting. We provide reliable techniques to detect such ectopic gene targeting which represents an unexpected caveat in mouse genetic engineering that should be considered in the design and validation strategy of future gene knock-in approaches.

Modeling Chromosomes in Mouse to Explore the Function of Genes, Genomic Disorders, and Chromosomal Organization

One of the challenges of genomic research after the completion of the human genome project is to assign a function to all the genes and to understand their interactions and organizations. Among the various techniques, the emergence of chromosome engineering tools with the aim to manipulate large genomic regions in the mouse model offers a powerful way to accelerate the discovery of gene functions and provides more mouse models to study normal and pathological developmental processes associated with aneuploidy. The combination of gene targeting in ES cells, recombinase technology, and other techniques makes it possible to generate new chromosomes carrying specific and defined deletions, duplications, inversions, and translocations that are accelerating functional analysis. This review presents the current status of chromosome engineering techniques and discusses the different applications as well as the implication of these new techniques in future research to better understand the function of chromosomal organization and structures.

Two ways to trap a gene in mice

Two ways to trap a gene in mice

A mouse model of tuberous sclerosis 1 showing background specific early post-natal mortality and metastatic renal cell carcinoma

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in either the TSC1 or the TSC2 genes and characterized by the development of benign hamartomatous growths in multiple organ systems. We have inactivated Tsc1 in the mouse germ line by gene targeting in ES cells and confirmed that the mutant allele (Tsc1-) has a recessive embryonic lethal phenotype. We found that a significant number (approximately 27%) of heterozygous (Tsc1+/-) mice on the C57BL/6 background died before weaning (P = 0.014) and show that these mice die in the post-natal period (P = 0.033), normally at 1-2 days, from unknown causes. Forty-four percent (7/16) of Tsc1+/- mice on a C3H background developed macroscopically visible renal lesions as early as 3-6 months, increasing to 95% (37/39) by 15-18 months. Renal lesions progressed from cysts through cystadenomas to solid carcinomas. Eighty percent (16/20) of Tsc1+/- mice on a Balb/c background exhibited solid renal cell carcinomas (RCC) by 15-18 months and in 41%, RCCs were > or = 5 mm, resulting in grossly deformed kidneys. Some RCCs had a sarcomatoid morphology of spindle cells in whorled patterns and metastasized to the lungs. We detected loss of the wild-type Tsc1 allele and elevated levels of p-mTOR and p-S6 in lesions from Tsc1+/- mice. This new murine model of hamartin deficiency exhibits a more severe phenotype than existing models.

Histone variant H2A.Z is required for early mammalian development

Fundamental to the process of mammalian development is the timed and coordinated regulation of gene expression. This requires transcription of a precise subset of the total complement of genes. It is clear that chromatin architecture plays a fundamental role in this process by either facilitating or restricting transcription factor binding [1]. How such specialized chromatin structures are established to regulate gene expression is poorly understood. All eukaryotic organisms contain specialized histone variants with distinctly different amino acid sequences that are even more conserved than the major core histones [2]. On the basis of their highly conserved sequence, histone variants have been assumed critical for the function of mammalian chromatin; however, a requirement for a histone variant has not been shown in mammalian cells. Mice with a deletion of H1° have been generated by gene targeting in ES cells, but these mice show no phenotypic consequences, perhaps due to redundancy of function [3]. Here we show for the first time that a mammalian histone variant, H2A.Z, plays a critical role in early development, and we conclude that this histone variant plays a pivotal role in establishing the chromatin structures required for the complex patterns of gene expression essential for normal mammalian development.

Knockout mice and steroid receptor research.

The development of techniques that allow defined alterations of the mammalian genome have dramatically increased the possibilities for elucidating functions of specific genes in the context of a whole organism. Although some of these techniques have been applied to several mammalian species, it is primarily the mouse that is the subject for these types of experiments. The first technique developed in this field was transgene technology, involving the incorporation of new copies of a gene into the genome of the host organism, and was successfully performed for the first time in 1980 (,). The other technique used to alter the mouse genome is referred to as “gene-targeting technology,” or more popularly, “knockout technology.” This technology involves homologous recombination in embryonic stem cells (ES cells) and was developed during the same decade (,) Gene targeting in ES cells has allowed scientists to perform studies of gene function in a way that has never been possible before. This review focuses on knockout technology, but also includes some background about transgenesis, because of its relevance for conditional knockouts. For more detailed information regarding establishment of transgenic mice, see ref. 16.

The mammalian Tolloid-like 1 gene, Tll1, is necessary for normal septation and positioning of the heart.

Mammalian Tolloid-like 1 (mTLL-1) is an astacin-like metalloprotease, highly similar in domain structure to the morphogenetically important proteases bone morphogenetic protein-1 (BMP-1) and Drosophila Tolloid. To investigate possible roles for mTLL-1 in mammalian development, we have used gene targeting in ES cells to produce mice with a disrupted allele for the corresponding gene, Tll1. Homozygous mutants were embryonic lethal, with death at mid-gestation from cardiac failure and a unique constellation of developmental defects that were apparently confined solely to the heart. Constant features were incomplete formation of the muscular interventricular septum and an abnormal and novel positioning of the heart and aorta. Consistent with roles in cardiac development, Tll1 expression was specific to precardiac tissue and endocardium in 7.5 and 8.5 days p.c. embryos, respectively. Tll1 expression was also high in the developing interventricular septum, where expression of the BMP-1 gene, Bmp1, was not observed. Cardiac structures that were not affected in Tll1-/- embryos either showed no Tll1 expression (atrio-ventricular cushions) or showed overlapping expression of Tll1 and Bmp1 (aortico-pulmonary septum), suggesting that products of the Bmp1 gene may be capable of functionally substituting for mTLL-1 at sites in which they are co-expressed. Together, the various data show that mTLL-1 plays multiple roles in formation of the mammalian heart and is essential for formation of the interventricular septum.

ApoA-I knockout mice: characterization of HDL metabolism in homozygotes and identification of a post-RNA mechanism of apoA-I up-regulation in heterozygotes

The major high density lipoprotein (HDL) apolipoprotein, apoA-I, was knocked out by gene targeting in ES cells to provide a model for the study of HDL metabolism and its relationship to plasma and tissue cholesterol metabolism. HDL and non-HDL cholesterol (HDL-C) were reduced in apoA-I-deficient mice. Feeding a high fat-high cholesterol diet raised HDL-C minimally in apoA-I knockout compared to the large increase seen in control mice, suggesting an interaction between diet and apoA-I genotype. In apoA-I-deficient mice, HDL was normal in size but altered in composition. Compared to control mice there was more triglyceride and free cholesterol and less cholesteryl ester (CE), suggesting that apoA-I-deficient HDL is a poor substrate for hepatic lipase and lecithin:cholesterol acyltransferase (LCAT). The metabolic basis of the low HDL-C levels in the apoA-I knockout mice was decreased flux into the HDL CE pool. The absolute delivery of HDL CE to both peripheral tissues and liver was also decreased. As tissue cholesterol levels and synthesis were unchanged, the decreased flux of cholesterol into the HDL CE pool was most likely due to decreased efflux of cholesterol from the peripheral tissues and decreased functional LCAT activity. The low HDL-C state in the apoA-I-deficient mouse was associated with an absolute decrease in unidirectional transport of cholesterol from peripheral tissues to the liver but this did not lead to cholesterol accumulation in the periphery or a cholesterol deficit in the liver; nor was there altered peripheral tissue HMG-CoA reductase activity. The only sign of decreased cholesterol flux to the liver was a 2.3-fold decrease in liver cholesterol 7 alpha-hydroxylase mRNA, suggesting decreased bile acid synthesis. In the apoA-I knockout mouse model it appears that low HDL levels create a new steady state in which decreased cholesterol is delivered to both peripheral tissues and the liver.

Gene targeting in ES cells.

Gene targeting in ES cells.

Gene Targeting in ES Cells

Gene Targeting in ES Cells

Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice.

Platelet-derived growth factor, a major mitogen and chemoattractant for a number of cell types, is implicated in the processes of wound healing, tumorigenesis, and differentiation and is recognized by two receptors, alpha and beta. To begin understanding the role of these receptors in development, beta-receptor-deficient mice were generated by gene targeting in ES cells. Mutant mice are hemorrhagic, thrombocytopenic, and severely anemic, exhibit a defect in kidney glomeruli because of a lack of mesangial cells, and die at or shortly before birth. However, many cell types and tissues that express the receptor, including major blood vessels and the heart, appear normal in the absence of the receptor. These results indicate that whereas the beta receptor is essential in certain cell types during embryonic development, its broader role may be masked because of compensation by the alpha-subunit.

Targeted disruption of the neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues.

The neurofibromatosis (NF1) gene shows significant homology to mammalian GAP and is an important regulator of the ras signal transduction pathway. To study the function of NF1 in normal development and to try and develop a mouse model of NF1 disease, we have used gene targeting in ES cells to generate mice carrying a null mutation at the mouse Nf1 locus. Although heterozygous mutant mice, aged up to 10 months, likely attributable to a severe malformation of the heart. Interestingly, mutant embryos also display hyperplasia of neural crest-derived sympathetic ganglia. These results identify new roles for NF1 in development and indicate that some of the abnormal growth phenomena observed in NF1 patients can be recapitulated in neurofibromin-deficient mice.

What the papers say: Mutation of N‐myc in Mice: What does the phenotype tell us?

Oncogenesis is manifested as uncontrolled cellular proliferation and in some situations a failure of normal differentiation in the transformed cell. This has led to speculation that the normal role of proto-oncogenes during development may be to mediate the relationship between proliferation and differentiation. The advent of gene targeting in ES cells allows the role oncogenes in development to be tested directly. Two recent studies have examined the phenotype of N-myc mutant mice generated by gene targeting. In both reports, the mutation is an embryonic lethal at 11.5 days of gestation confirming a critical role for this proto-oncogene in development and the inability of other members of the myc family to substitute functionally for N-myc. Although the phenotypes are similar in general outline, the two reports differ in the specifics of the morphological and histological abnormalities identified. The disparity may result from the mutation created, the genetic background of the mutant mice or the criteria used to determine abnormalities. Assuredly, there is valuable information to be gained about N-myc function from these mutant mice. However, these reports make it clear that morphological and histological abnormalities in N-myc mutant mice serve as a starting point rather than as an endpoint. The challenge now is to link the defect at the cellular level to the abnormalities at the physiological level.