Human BAC-mediated rescue of the Friedreich ataxia knockout mutation in transgenic mice

Three independent transgenic mouse lines were generated with the human Friedreich ataxia gene, FRDA, in an 188-kb bacterial artificial chromosome (BAC) genomic sequence. Three copies of the transgene per diploid mouse genome were integrated in a single site in each mouse line. Transgenic mice were mated with mice heterozygous for a knockout mutation of the murine Frda gene, to generate mice homozygous for the Frda knockout mutation and hemizygous or homozygous for the human transgene. Rescue of the embryonic lethality that is associated with homozygosity for the Frda knockout mutation was observed in all three lines. Rescued mice displayed normal behavioral and biochemical parameters. RT-PCR analysis demonstrated that human FRDA mRNA is expressed in all the lines. The relative expression of the human FRDA and mouse Frda genes showed a similar pattern in different tissues in all three lines, indicating position-independent control of expression of the human FRDA transgene. However, large differences in the human:mouse mRNA ratio were observed between different tissues in all three lines. The human transgene is expressed at much higher levels in the brain, liver, and skeletal muscle than the endogenous gene, while expression of the human transgene in blood is only 25–30% of the mouse gene. These studies will facilitate the development of humanized mouse models of Friedreich ataxia through introduction of a GAA trinucleotide expansion or specific known point mutations in the normal human FRDA locus and the study of the regulation of gene expression from the FRDA locus.     

Cre-loxP-mediated gene replacement: a mouse strain producing humanized antibodies

Background: The bacteriophage-derived Cre–loxP recombination system operates efficiently in mammalian cells. This system is particularly useful in gene-targeting experiments in the mouse, and has already been used to generate ‘clean’ deletions of target genes in the germ line, as well as to inactivate target genes in a conditional manner (based on regulated expression of the Cre recombinase). In principle, Cre–loxP-mediated recombination should also allow gene replacement, and thus the introduction of virtually any kind of mutation into the genome.Results We used the Cre–loxP system, in mouse embryonic stem cells, to replace the mouse gene Cγ 1, which encodes the constant region of the heavy chain of IgG1 antibodies, with its human counterpart. The mutation was transmitted through the mouse germ line, and the resulting mutant mice were crossed with mice expressing κ light chains with a human, instead of a mouse, constant region. Mice homozygous for both mutations produce humanized, κ-chain-bearing IgG1 antibodies at the same level and efficiency as wild-type mice produce murine IgG1 antibodies. These animals should enable the ex vivo production of humanized, chimeric monoclonal antibodies specific for any antigen to which the mouse can respond.Conclusion Cre–loxP-mediated gene replacement is a simple and efficient general method of targeted mutagenesis in the mouse.     

A BACwards glance at neurodegeneration: molecular insights into disease from LRRK2, SNCA and MAPT BAC-transgenic mice

BAC (bacterial artificial chromosome)-transgenic mice expressing a transgene from an entire genomic locus under the control of the native promoter offer the opportunity to generate more accurate genetic models of human disease. The present review discusses results of recent studies investigating PD (Parkinson's disease) and tauopathies using BAC-transgenic mice carrying either the LRRK2 (leucine-rich repeat kinase 2), α-synuclein (SNCA) or MAPT (microtubule-associated protein tau) genes. In all lines, expression of the WT (wild-type) gene resulted in physiologically relevant protein expression. The effect of expressing the mutant form of a gene varied depending on the mouse strain or the particular disease mutation used, although it was common to see either neurochemical or behavioural differences in these animals. Overall, BAC technology offers an exciting opportunity to generate a wide range of new animal models of human-disease states.     

Expression of HLA class II molecules in humanized NOD.Rag1KO.IL2RgcKO mice is critical for development and function of human T and B cells

Background

Humanized mice able to reconstitute a surrogate human immune system (HIS) can be used for studies on human immunology and may provide a predictive preclinical model for human vaccines prior to clinical trials. However, current humanized mouse models show sub-optimal human T cell reconstitution and limited ability to support immunoglobulin class switching by human B cells. This limitation has been attributed to the lack of expression of Human Leukocyte Antigens (HLA) molecules in mouse lymphoid organs. Recently, humanized mice expressing HLA class I molecules have been generated but showed little improvement in human T cell reconstitution and function of T and B cells.

Methods

We have generated NOD.Rag1KO.IL2RγcKO mice expressing HLA class II (HLA-DR4) molecules under the I-E^d promoter that were infused as adults with HLA-DR-matched human hematopoietic stem cells (HSC). Littermates lacking expression of HLA-DR4 molecules were used as control.

Results

HSC-infused HLA-DR4.NOD.Rag1KO.IL-2RγcKO mice developed a very high reconstitution rate (>90%) with long-lived and functional human T and B cells. Unlike previous humanized mouse models reported in the literature and our control mice, the HLA-DR4 expressing mice reconstituted serum levels (natural antibodies) of human IgM, IgG (all four subclasses), IgA, and IgE comparable to humans, and elicited high titers of specific human IgG antibodies upon tetanus toxoid vaccination.

Conclusions

Our study demonstrates the critical role of HLA class II molecules for development of functional human T cells able to support immunoglobulin class switching and efficiently respond to vaccination.     

Humanized c-Myc Mouse

Background

A given tumor is usually dependent on the oncogene that is activated in the respective tumor entity. This phenomenon called oncogene addiction provides the rationale for attempts to target oncogene products in a therapeutic manner, be it by small molecules, by small interfering RNAs (siRNA) or by antigen-specific T cells. As the proto-oncogene product is required also for the function of normal cells, this raises the question whether there is a therapeutic window between the adverse effects of specific inhibitors or T cells to normal tissue that may limit their application, and their beneficial tumor-specific therapeutic action. To address this crucial question, suitable mouse strains need to be developed, that enable expression of the human proto-oncogene not only in tumor but also in normal cells. The aim of this work is to provide such a mouse strain for the human proto-oncogene product c-MYC.

Principal Findings

We generated C57BL/6-derived embryonic stem cells that are transgenic for a humanized c-Myc gene and established a mouse strain (hc-Myc) that expresses human c-MYC instead of the murine ortholog. These transgenic animals harbor the humanized c-Myc gene integrated into the endogenous murine c-Myc locus. Despite the lack of the endogenous murine c-Myc gene, homozygous mice show a normal phenotype indicating that human c-MYC can replace its murine ortholog.

Conclusions

The newly established hc-Myc mouse strain provides a model system to study in detail the adverse effects of therapies that target the human c-MYC protein. To mimic the clinical situation, hc-Myc mice may be cross-bred to mice that develop tumors due to overexpression of human c-MYC. With these double transgenic mice it will be possible to study simultaneously the therapeutic efficiency and adverse side effects of MYC-specific therapies in the same mouse.     

Association of MHC and rheumatoid arthritis: HLA-DR4 and rheumatoid arthritis - studies in mice and men

Inherited susceptibility to rheumatoid arthritis (RA) is associated with the DRB1 genes encoding the human leukocyte antigen (HLA)-DR4 and HLA-DR1 molecules. Transgenic mice expressing these major histocompatibility complex (MHC) class II molecules have been developed to generate humanized models for RA. The relevance of these models for understanding RA will be discussed.     

Association of MHC and rheumatoid arthritis. HLA-DR4 and rheumatoid arthritis: studies in mice and men

Inherited susceptibility to rheumatoid arthritis (RA) is associated with the DRB1 genes encoding the human leukocyte antigen (HLA)-DR4 and HLA-DR1 molecules. Transgenic mice expressing these major histocompatibility complex (MHC) class II molecules have been developed to generate humanized models for RA. The relevance of these models for understanding RA will be discussed.     

Effects of BRCA1 transgene expression on murine mammary gland development and mutagen-induced mammary neoplasia

To characterize the role of BRCA1 in mammary gland development and tumor suppression, a transgenic mouse model of BRCA1 overexpression was developed. Using the mouse mammary tumor virus (MMTV) promoter/enhancer, transgenic mice expressing human BRCA1 or select mutant controls were generated. Transgenic animals examined during adolescence were shown to express the human transgene in their mammary glands. The mammary glands of 13-week-old virgin homozygous MMTV-BRCA1 mice presented the morphology of moderately increased lobulo-alveolar development. The mammary ductal trees of both hemizygous and homozygous MMTV-BRCA1t340 were similar to those of control non-transgenic littermates. Interestingly, both hemi- and homozygous mice expressing a splice variant of BRCA1 lacking the N-terminal RING finger domain (MMTV-BRCA1sv) exhibited marked mammary lobulo-alveolar development, particularly terminal end bud proliferation. Morphometric analyses of mammary gland whole mount preparations were used to measure epithelial staining indices of ~35% for homozygous MMTV-BRCA1 mice and ~60% for both hemizygous and homozygous MMTV-BRCA1sv mice versus ~25% for non-transgenic mice. Homozygous MMTV-BRCA1 mice showed delayed development of tumors when challenged with 7,12 dimethylbenzanthracene (DMBA), relative to non-transgenic and homozygous BRCA1t340 expressing mice. In contrast, homozygous MMTV-BRCA1sv transgenic animals were sensitized to DMBA treatment and exhibited a very rapid onset of mammary tumor development and accelerated mortality. MMTV-BRCA1 effects on mortality were restricted to DMBA-induced tumors of the mammary gland. These results demonstrate in vivo roles for BRCA1 in both mammary gland development and in tumor suppression against mutagen-induced mammary gland neoplasia.     

The reconstituted ‘humanized liver’ in TK-NOG mice is mature and functional

To overcome the limitations of existing models, we developed a novel experimental in vivo platform for replacing mouse liver with functioning human liver tissue. To do this, a herpes simplex virus type 1 thymidine kinase (HSVtk) transgene was expressed within the liver of highly immunodeficient NOG mice (TK-NOG). Mouse liver cells expressing this transgene were ablated after a brief exposure to a non-toxic dose of ganciclovir (GCV), and transplanted human liver cells are stably maintained within the liver (humanized TK-NOG) without exogenous drug. The reconstituted liver was shown to be a mature and functioning “human organ” that had zonal position-specific enzyme expression and a global gene expression pattern representative of mature human liver; and could generate a human-specific profile of drug metabolism. The ‘humanized liver’ could be stably maintained in these mice with a high level of synthetic function for a prolonged period (8 months). This novel in vivo system provides an optimized platform for studying human liver physiology, including drug metabolism, toxicology, or liver regeneration.     

Generation of a mouse expressing a conditional knockout of the hepatocyte growth factor gene: demonstration of impaired liver regeneration

Hepatocyte growth/scatter factor (HGF/SF) is a pleiotropic cytokine originally identified as a potent mitogen for rat hepatocytes. Two HGF/SF knockout mouse models have been reported, both of which exhibit developmental abnormalities causing embryonic lethality. To circumvent this limitation, we created a mouse conditionally deficient in liver expression of HGF/SF to specifically investigate the role of this mitogen in the process of adult liver regeneration. Gene targeting technology was used to generate a mouse with loxP sites flanking exon 5 of the HGF/SF gene (ex5-flox). In the absence of cre recombinase activity, mice homozygous for ex5-flox were indistinguishable from wild-type littermates. To ablate HGF/SF gene expression in vitro, primary hepatocytes established from homozygous HGF(ex5-flox) mice were infected with a recombinant adenoviral vector coding for cre recombinase (AdCre1). PCR analyses of genomic DNA demonstrated greater than 90% ablation of the ex5-floxed gene sequence. In vivo, HGF(ex.5-flox) mice were administered AdCre1 vector and the ablation of the HGF gene confirmed by Southern blot analysis. To induce liver regeneration, mice were injected with the hepatotoxin carbon tetrachloride. The regenerative capacity of hepatocytes in mice administered cre recombinase was shown to be significantly reduced when compared with mice injected with an adenovirus expressing LacZ. A similar reduction in hepatocyte regeneration was observed in HGF(ex.5.flox) mice carrying the cre transgene under the control of the interferon-inducible (pI:pC) Mx1 promoter, as an alternative strategy to ablate the HGF/SF gene in liver. Our results confirm the mitogenic role of HGF/SF in liver regeneration.     

Complete functional rescue of the ABCA1-/- mouse by human BAC transgenesis

Humanized mouse models are useful tools to explore the functional and regulatory differences between human and murine orthologous genes. We have combined a bioinformatics approach and an in vivo approach to assess the functional and regulatory differences between the human and mouse ABCA1 genes. Computational analysis identified significant differences in potential regulatory sites between the human and mouse genes. The effect of these differences was assessed in vivo, using a bacterial artificial chromosome transgenic humanized ABCA1 mouse model that expresses the human gene in the absence of mouse ABCA1. Humanized mice expressed human ABCA1 protein at levels similar to wild-type mice and fully compensated for cholesterol efflux activity and lipid levels seen in ABCA1-deficient mice. Liver X receptor agonist administration resulted in significant increases in HDL values associated with parallel increases in the hepatic ABCA1 protein and mRNA levels in the humanized ABCA1 mice, as seen in the wild-type animals. Our studies indicate that despite differences in potential regulatory regions, the human ABCA1 gene is able to functionally fully compensate for the mouse gene. Our humanized ABCA1 mice can serve as a useful model system for functional analysis of the human ABCA1 gene in vivo and can be used for the generation of potential new therapeutics that target HDL metabolism.     

Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rγnull mouse model

Purpose  There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities. Thus, the first aim of this study was to establish a human tumor-bearing mouse model in which human immune cells can engraft and mediate ADCC, but where the endogenous mouse immune cells cannot mediate ADCC. The second aim was to evaluate ADCC mediated in these humanized mice by the defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody (mAb) which we have developed and which is now in phase I clinical trials. Experimental design  NOD/Shi-scid, IL-2Rγ^null (NOG) mice were the recipients of human immune cells, and CCR4-expressing Hodgkin lymphoma (HL) and cutaneous T-cell lymphoma (CTCL) cell lines were used as target tumors. Results  Humanized mice have been established using NOG mice. The chimeric defucosylated anti-CCR4 mAb KM2760 showed potent antitumor activity mediated by robust ADCC in these humanized mice bearing the HL or CTCL cell lines. KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in HL-bearing humanized mice. Conclusions  Anti-CCR4 mAb could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. In addition, using our humanized mice, we can perform the appropriate preclinical evaluation of many types of antibody based immunotherapy.     

Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice

We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression—not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies.     

Human BRCA1 gene rescues the embryonic lethality of Brca1 mutant mice

Half of all familial breast cancers are due to mutation in the BRCA1 gene. However, despite its importance, attempts to model BRCA1-induced disease in the mouse have been disappointing. Heterozygous Brca1 knockout mice do not develop mammary tumors and homozygous knockout mice die during embryogenesis from ill-defined causes. Sequence analysis has shown that the coding region, genomic organization, and regulatory sequences of the human and mouse genes are not well conserved. This has raised the question of whether the mouse can serve as an effective model for functional analysis of the human BRCA1 gene. To address this question we have introduced a bacterial artificial chromosome containing the human BRCA1 gene into the germline of Brca1 knockout mice. Surprisingly, we have found that the embryonic lethality of Brca1 knockout mice is rescued by the human transgene. We also show that expression of human BRCA1 transgene mirrors the endogenous murine gene. Our "humanized" transgenic mice can serve as a model system for functional analyses of the human BRCA1 gene. Published 2001 Wiley-Liss, Inc.     

CYP3A4 and pregnane X receptor humanized mice

Marked species differences exist in P450 expression and activities. In order to produce mouse models that can be used to more accurately predict human drug and carcinogen metabolism, P450- and xenobiotic receptor humanized mice are being prepared using bacterial artificial chromosomes (BAC) and P1 phage artificial chromosomes (PAC) genomic clones. In some cases, transgenic mice carrying the human genes are bred with null-mice to produce fully humanized mice. Mice expressing human CYP1A1, CYP1A2, CYP2E1, CYP2D6, CYP3A4, and CYP3A7 were generated and characterized. Studies with the CYP3A4-humanized (hCYP3A4) mouse line revealed new information on the physiological function of this P450 and its role in drug metabolism in vivo. With this mouse line, CYP3A4, under certain circumstances, was found to alter the serum levels of estrogen resulting in deficient lactation and low pup survival as a result of underdeveloped mammary glands. This hCYP3A4 mouse established the importance of intestinal CYP3A4 in the pharmacokinetics of orally administered drugs. The hCYP3A4 mice were also used to establish the mechanisms of potential gender differences in CYP3A4 expression (adult female > adult male) that could account for human gender differences in drug metabolism and response. The pregnane X receptor (PXR) is also involved in induction of drug metabolism through its target genes including CYP3A4. Since species differences exist in ligand specificity between human and mice, a PXR-humanized mouse (hPXR) was produced that responds to human PXR activators such as rifampicin but does not respond to the rodent activator pregnenalone 16alpha-carbonitrile.     

The First Cellular Models Based on Frataxin Missense Mutations That Reproduce Spontaneously the Defects Associated with Friedreich Ataxia

Background

Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)_n expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation.

Methodology

We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN^G130V and hFXN^I154F) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN^I154F mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients.

Conclusions

These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency.     

The Clouston syndrome mutation connexin30 A88V leads to hyperproliferation of sebaceous glands and hearing impairments in mice

Distinct mutations in the gap junction protein connexin30 (Cx30) can cause the ectodermal dysplasia Clouston syndrome in humans. We have generated a new mouse line expressing the Clouston syndrome mutation Cx30A88V under the control of the endogenous Cx30 promoter. Our results show that the mutated Cx30A88V protein is incorporated in gap junctional plaques of the epidermis. Homozygous Cx30A88V mice reveal hyperproliferative and enlarged sebaceous glands as well as a mild palmoplantar hyperkeratosis. Additionally, homozygous mutant mice show an altered hearing profile compared to control mice. We conclude that the Cx30A88V mutation triggers hyperproliferation in the skin and changes the cochlear homeostasis in mice.     

Mouse models with human immunity and their application in biomedical research

Biomedical research in human beings is largely restricted to in vitro studies that lack complexity of a living organism. To overcome this limitation, humanized mouse models are developed based on immunodeficient characteristics of severe combined immunodeficiency (SCID) or recombination activating gene (Rag)^null mice, which can accept xenografts. Peripheral constitution of human immunity in SCID or Rag^null mice has been achieved by transplantation of mature human immune cells, foetal human thymus, bone marrow, liver tissues, lymph nodes or a combination of these, although efficiency needs to be improved. These mouse models with constituted human immunity (defined as humanized mice in the present text) have been widely used to investigate the basic principles of human immunobiology as well as complex pathomechanisms and potential therapies of human diseases. Here, elements of an ideal humanized mouse model are highlighted including genetic and non-genetic modification of recipient mice, transplantation strategies and proposals to improve engraftments. The applications of the humanized mice to study the development and response of human immune cells, human autoimmune diseases, virus infections, transplantation biology and tumour biology are reviewed as well.     

APP mouse models for Alzheimer's disease preclinical studies

Animal models of human diseases that accurately recapitulate clinical pathology are indispensable for understanding molecular mechanisms and advancing preclinical studies. The Alzheimer's disease (AD) research community has historically used first‐generation transgenic (Tg) mouse models that overexpress proteins linked to familial AD (FAD), mutant amyloid precursor protein (APP), or APP and presenilin (PS). These mice exhibit AD pathology, but the overexpression paradigm may cause additional phenotypes unrelated to AD. Second‐generation mouse models contain humanized sequences and clinical mutations in the endogenous mouse App gene. These mice show Aβ accumulation without phenotypes related to overexpression but are not yet a clinical recapitulation of human AD. In this review, we evaluate different APP mouse models of AD, and review recent studies using the second‐generation mice. We advise AD researchers to consider the comparative strengths and limitations of each model against the scientific and therapeutic goal of a prospective preclinical study.