Bacterial Artificial Chromosome Transgenesis Research Articles

Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes

Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. We generated 707 transgenic founders from 86 BAC transgenes purified by three different methods. Transgenesis efficiency was the same for all BAC DNA purification methods. Polyamine microinjection buffer was essential for successful integration of intact BAC transgenes. There was no correlation between BAC size and transgenic rate, birth rate, or transgenic efficiency. A narrow DNA concentration range generated the best transgenic efficiency. High DNA concentrations reduced birth rates while very low concentrations resulted in higher birth rates and lower transgenic efficiency. Founders with complete BAC integrations were observed in all 47 BACs for which multiple markers were tested. Additional founders with BAC fragment integrations were observed for 65% of these BACs. Expression data was available for 79 BAC transgenes and expression was observed in transgenic founders from 63 BACs (80%). Consistent and reproducible success in BAC transgenesis required the combination of careful DNA purification, the use of polyamine buffer, and sensitive genotyping assays.

BAC Transgenesis in Human Embryonic Stem Cells as a Novel Tool to Define the Human Neural Lineage

Human embryonic stem cells (hESCs) have enormous potential for applications in basic biology and regenerative medicine. However, harnessing the potential of hESCs toward generating homogeneous populations of specialized cells remains challenging. Here we describe a novel technology for the genetic identification of defined hESC-derived neural cell types using bacterial artificial chromosome (BAC) transgenesis. We generated hESC lines stably expressing Hes5::GFP, Dll1::GFP, and HB9::GFP BACs that yield green fluorescent protein (GFP)(+) neural stem cells, neuroblasts, and motor neurons, respectively. Faithful reporter expression was confirmed by cell fate analysis and appropriate transgene regulation. Prospective isolation of HB9::GFP(+) cells yielded purified human motor neurons with proper marker expression and electrophysiological activity. Global mRNA and microRNA analyses of Hes5::GFP(+) and HB9::GFP(+) populations revealed highly specific expression signatures, suggesting that BAC transgenesis will be a powerful tool for establishing expression libraries that define the human neural lineage and for accessing defined cell types in applications of human disease.

Mice With Hyperghrelinemia Are Hyperphagic and Glucose Intolerant and Have Reduced Leptin Sensitivity

OBJECTIVEGhrelin is the only known peripheral hormone to increase ingestive behavior. However, its role in the physiological regulation of energy homeostasis is unclear because deletion of ghrelin or its receptor does not alter food intake or body weight in mice fed a normal chow diet. We hypothesized that overexpression of ghrelin in its physiological tissues would increase food intake and body weight.RESEARCH DESIGN AND METHODSWe used bacterial artificial chromosome transgenesis to generate a mouse model with increased ghrelin expression and production in the stomach and brain. We investigated the effect of ghrelin overexpression on food intake and body weight. We also measured energy expenditure and determined glucose tolerance, glucose stimulated insulin release, and peripheral insulin sensitivity.RESULTSGhrelin transgenic (Tg) mice exhibited increased circulating bioactive ghrelin, which was associated with hyperphagia, increased energy expenditure, glucose intolerance, decreased glucose stimulated insulin secretion, and reduced leptin sensitivity.CONCLUSIONSThis is the first report of a Tg approach suggesting that ghrelin regulates appetite under normal feeding conditions and provides evidence that ghrelin plays a fundamental role in regulating β-cell function.

A Double Transgenic Mouse Model Expressing Human Pregnane X Receptor and Cytochrome P450 3A4

Cytochrome P450 3A4 (CYP3A4), the most abundant human cytochrome P450 in liver, participates in the metabolism of approximately 50% of clinically used drugs. The pregnane X receptor (PXR), a member of the nuclear receptor superfamily, is the major activator of CYP3A4 transcription. However, because of species differences in response to PXR ligands, it is problematic to use rodents to assess CYP3A4 regulation and function. The generation of double transgenic mice expressing human PXR and CYP3A4 (TgCYP3A4/hPXR) would provide a solution to this problem. In the current study, a TgCYP3A4/hPXR mouse model was generated by bacterial artificial chromosome transgenesis in Pxr-null mice. In TgCYP3A4/hPXR mice, CYP3A4 was strongly induced by rifampicin, a human-specific PXR ligand, but not by pregnenolone 16alpha-carbonitrile, a rodent-specific PXR ligand. Consistent with CYP3A expression, hepatic CYP3A activity increased approximately 5-fold in TgCYP3A4/hPXR mice pretreated with rifampicin. Most antihuman immunodeficiency virus protease inhibitors are CYP3A substrates and their interactions with rifamycins are a source of major concern in patients coinfected with human immunodeficiency virus and Mycobacterium tuberculosis. By using TgCYP3A4/hPXR mice, human PXR-CYP3A4-mediated rifampicin-protease inhibitor interactions were recapitulated, as the metabolic stability of amprenavir, nelfinavir, and saquinavir decreased 52, 53, and 99%, respectively, in the liver microsomes of TgCYP3A4/hPXR mice pretreated with rifampicin. In vivo, rifampicin pretreatment resulted in an approximately 80% decrease in the area under the serum amprenavir concentration-time curve in TgCYP3A4/hPXR mice. These results suggest that the TgCYP3A4/hPXR mouse model could serve as a useful tool for studies on CYP3A4 transcription and function in vivo.

Enriched Motor Neuron Populations Derived from Bacterial Artificial Chromosome-transgenic Human Embryonic Stem Cells

INTRODUCTION: Human embryonic stem cells (hESCs) can produce somatic cells of all three germ layers. The ability to obtain enriched populations of hESC-derived neurons is essential to their utilization toward cell replacement therapy and understanding human disease. Here we describe a new technology that allows the transgenesis of hESCs with a bacterial artificial chromosome (BAC) expressing green fluorescent protein (GFP) under the control of the motor neuron-specific Hb9 promoter. METHODS: Human ESCs were stably transfected with the Hb9:GFP BAC via nucleofection. Of 28 transfected clones, one (3.6%) expressed GFP in motor neurons. The clone had a normal karyotype and FISH analysis indicated a single BAC integration site at chromosome 7q31. RESULTS: The GFP-positive motor neurons expressed the transcription factors Hb9, Isl1, Lim3 and Nkx6.1, all relevant to the motor neuron lineage, and were postmitotic. Using flow cytometry, we were able to enrich the GFP-positive motor neuron pool and culture it long-term on hESC-derived mesenchymal feeders. Transcriptome analysis using microarray technology revealed 78 transcripts upregulated in motor neurons compared to neural precursors and 49 transcripts upregulated compared with GFP-negative cells. The majority of these transcripts have not been described before in motor neurons. Ongoing studies include electrophysiological recordings from these motor neurons, expressing mutant superoxide dismutase 1 (mSOD1) to generate a human Amyotrophic Lateral Sclerosis model, and transplanting them into healthy and mSOD1-transgenic rats to test their survival and engraftment potential in vivo. CONCLUSION: We have developed a novel technology involving the BAC transgenesis of hESCs. BAC promoters confer a much higher fidelity of transgene expression than conventional short promoters. We have used this technology to generate transgenic hESCs that produce GFP-positive motor neurons. Such motor neurons can be enriched by flow cytometry and used toward transcriptome analysis, disease model generation, and transplantation studies.

Intrinsic and Extrinsic Light Responses in Melanopsin-Expressing Ganglion Cells During Mouse Development

Melanopsin (Opn4) is a photopigment found in a subset of retinal ganglion cells (RGCs) that project to various brain areas. These neurons are intrinsically photosensitive (ipRGCs) and are implicated in nonimage-forming responses to environmental light such as the pupillary light reflex and circadian entrainment. Recent evidence indicates that ipRGCs respond to light at birth, but questions remain as to whether and when they undergo significant functional changes. We used bacterial artificial chromosome transgenesis to engineer a mouse line in which enhanced green fluorescent protein (EGFP) is expressed under the control of the melanopsin promoter. Double immunolabeling for EGFP and melanopsin demonstrates their colocalization in ganglion cells of mutant mouse retinas. Electrophysiological recordings of ipRGCs in neonatal mice (postnatal day 0 [P0] to P7) demonstrated that these cells responded to light with small and sluggish depolarization. However, starting at P11 we observed ipRGCs that responded to light with a larger and faster onset (<1 s) and offset (<1 s) depolarization. These faster, larger depolarizations were observed in most ipRGCs by early adult ages. However, on application of a cocktail of synaptic blockers, we found that all cells responded to light with slow onset (>2.5 s) and offset (>10 s) depolarization, revealing the intrinsic, melanopsin-mediated light responses. The extrinsic, cone/rod influence on ipRGCs correlates with their extensive dendritic stratification in the inner plexiform layer. Collectively, these results demonstrate that ipRGCs make use of melanopsin for phototransduction before eye opening and that these cells further integrate signals derived from the outer retina as the retina matures.

Complex Role of the Mitochondrial Targeting Signal in the Function of Steroidogenic Acute Regulatory Protein Revealed by Bacterial Artificial Chromosome Transgenesis in Vivo

The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads. Thereafter, we targeted expression of either wild-type StAR (WT-StAR) or a mutated StAR protein lacking the mitochondrial targeting signal (N47-StAR). In the context of mice homozygous for a StAR knockout allele (StAR-/-), all StAR activity derived from the StAR transgenes, allowing us to examine the function of the proteins that they encode. The WT-StAR transgene consistently restored viability and steroidogenic function to StAR-/- mice. Although the N47-StAR protein was reportedly active in transfected COS cells and mitochondrial reconstitution experiments, the N47-StAR transgene rescued viability in only 40% of StAR-/- mice. Analysis of lipid deposits in the primary steroidogenic tissues revealed a hierarchy of StAR function provided by N47-StAR: florid lipid deposits were seen in the adrenal cortex and ovarian theca region, with milder deposits in the Leydig cells. Our results confirm the ability of StAR lacking its mitochondrial targeting signal to perform some essential functions in vivo but also demonstrate important functional defects that differ from in vitro studies obtained in nonsteroidogenic cells.

Molecular regulation of endocardial heterogeneity during semilunar valve development and disease

A critical event in cardiac morphogenesis is differentiation of heterogenous endocardial subpopulations with distinctive developmental capabilities. However, little is known about the factors that regulate endocardial differentiation. We have utilized NFATc‐Cre mediated lineage tracing in vivo to identify a subpopulation of valvular endocardial cells within the outflow tract that is resistant to transformation and is required for semilunar valve formation. Bacterial artificial chromosome (BAC) transgenesis containing endogenous regulatory elements of the NFATc1 locus was used to study the earliest events of endocardial differentiation. These studies have defined the endocardium as a unique subpopulation of endothelium during early mesodermal differentiation. Further analysis of in vitro endocardium differentiation utilizing ES cells derived from transgenic animals confirms that in vitro differentiation mirrors the temporal and spatial organization seen in vivo. Furthermore, manipulation of tissue specific culture conditions suggests that endocardial differentiation is not dependent on vascular or hematopoietic differentiation but is more closely associated and regulated by myocardial differentiation in a Wnt dependent process. These studies will be discussed in the context of developing a renewable resource of endocardial cells as mediators of tissue regeneration and repair.

Mosaic complementation demonstrates a regulatory role for myosin VIIa in actin dynamics of stereocilia.

We have developed a bacterial artificial chromosome transgenesis approach that allowed the expression of myosin VIIa from the mouse X chromosome. We demonstrated the complementation of the Myo7a null mutant phenotype producing a fine mosaic of two types of sensory hair cells within inner ear epithelia of hemizygous transgenic females due to X inactivation. Direct comparisons between neighboring auditory hair cells that were different only with respect to myosin VIIa expression revealed that mutant stereocilia are significantly longer than those of their complemented counterparts. Myosin VIIa-deficient hair cells showed an abnormally persistent tip localization of whirlin, a protein directly linked to elongation of stereocilia, in stereocilia. Furthermore, myosin VIIa localized at the tips of all abnormally short stereocilia of mice deficient for either myosin XVa or whirlin. Our results strongly suggest that myosin VIIa regulates the establishment of a setpoint for stereocilium heights, and this novel role may influence their normal staircase-like arrangement within a bundle.

Selective Loss of Leptin Receptors in the Ventromedial Hypothalamic Nucleus Results in Increased Adiposity and a Metabolic Syndrome

Leptin, an adipocyte-derived hormone, has emerged as a critical regulator of energy homeostasis. The leptin receptor (Lepr) is expressed in discrete regions of the brain; among the sites of highest expression are several mediobasal hypothalamic nuclei known to play a role in energy homeostasis, including the arcuate nucleus, the ventromedial hypothalamic nucleus (VMH), and the dorsomedial hypothalamic nucleus. Although most studies have focused on leptin's actions in the arcuate nucleus, the role of Lepr in these other sites has received less attention. To explore the role of leptin signaling in the VMH, we used bacterial artificial chromosome transgenesis to target Cre recombinase to VMH neurons expressing steroidogenic factor 1, thereby inactivating a conditional Lepr allele specifically in steroidogenic factor 1 neurons of the VMH. These knockout (KO) mice, designated Lepr KO(VMH), exhibited obesity, particularly when challenged with a high-fat diet. On a low-fat diet, Lepr KO(VMH) mice exhibited significantly increased adipose mass even when their weights were comparable to wild-type littermates. Furthermore, these mice exhibited a metabolic syndrome including hepatic steatosis, dyslipidemia, and hyperleptinemia. Lepr KO(VMH) mice were hyperinsulinemic from the age of weaning and eventually developed overt glucose intolerance. These data define nonredundant roles of the Lepr in VMH neurons in energy homeostasis and provide a model system for studying other actions of leptin in the VMH.

Dkk3‐Cre BAC transgenic mouse line: a tool for highly efficient gene deletion in retinal progenitor cells

To establish the genetic tools for conditional gene deletion in mouse retinal progenitors, we generated a Dkk3-Cre transgenic mouse line using bacterial artificial chromosome (BAC) transgenesis. Cre recombination efficiency in vivo was assayed by crossing this transgenic line, termed BAC-Dkk3-Cre, with the CAG-CAT-Z reporter line. This BAC-Dkk3-Cre line showed Cre recombinase activity in most retinal progenitors. Cre activity was detectable from embryonic day 10.5 (E10.5) and generally restricted to the retina during embryogenesis. To verify that BAC-Dkk3-Cre mice successfully circumvented lethality, we generated Otx2flox/flox/BAC-Dkk3-Cre+ mice as Otx2 conditional knockout mice. The Otx2flox/flox/BAC-Dkk3-Cre+ mice were viable, and their retina showed loss of mature cell-type markers of photoreceptor cells, bipolar cells, and horizontal cells, in contrast, amacrine-like cells noticeably increased. Thus, the BAC-Dkk3-Cre transgenic mouse line provides a powerful tool for generating conditional knockout mouse lines for studying loss of gene functions in the developing retina.

Physiologically regulated transgenic ABCA1 does not reduce amyloid burden or amyloid-β peptide levels in vivo

ABCA1-deficient mice have low levels of poorly lipidated apolipoprotein E (apoE) and exhibit increased amyloid load. To test whether excess ABCA1 protects from amyloid deposition, we crossed APP/PS1 mice to ABCA1 bacterial artificial chromosome (BAC) transgenic mice. Compared with wild-type animals, the ABCA1 BAC led to a 50% increase in cortical ABCA1 protein and a 15% increase in apoE abundance, demonstrating that this BAC supports modest ABCA1 overexpression in brain. However, this was observed only in animals that do not deposit amyloid. Comparison of ABCA1/APP/PS1 mice with APP/PS1 controls revealed no differences in levels of brain ABCA1 protein, amyloid, Abeta, or apoE, despite clear retention of ABCA1 overexpression in the livers of these animals. To further investigate ABCA1 expression in the amyloid-containing brain, we then compared ABCA1 mRNA and protein levels in young and aged cortex and cerebellum of APP/PS1 and ABCA1/APP/PS1 animals. Compared with APP/PS1 controls, aged ABCA1/APP/PS1 mice exhibited increased ABCA1 mRNA, but not protein, selectively in cortex. Additionally, ABCA1 mRNA levels were not increased before amyloid deposition but were induced only in the presence of extensive Abeta and amyloid levels. These data suggest that an induction of ABCA1 expression may be associated with late-stage Alzheimer's neuropathology.

The Pregnane X Receptor Gene-Humanized Mouse: A Model for Investigating Drug-Drug Interactions Mediated by Cytochromes P450 3A

The most common clinical implication for the activation of the human pregnane X receptor (PXR) is the occurrence of drug-drug interactions mediated by up-regulated cytochromes P450 3A (CYP3A) isozymes. Typical rodent models do not predict drug-drug interactions mediated by human PXR because of species differences in response to PXR ligands. In the current study, a PXR-humanized mouse model was generated by bacterial artificial chromosome (BAC) transgenesis in Pxr-null mice using a BAC clone containing the complete human PXR gene and 5'- and 3'-flanking sequences. In this PXR-humanized mouse model, PXR is selectively expressed in the liver and intestine, the same tissue expression pattern as CYP3A. Treatment of PXR-humanized mice with the PXR ligands mimicked the human response, since both hepatic and intestinal CYP3As were strongly induced by rifampicin, a human-specific PXR ligand, but not by pregnenolone 16alpha-carbonitrile, a rodent-specific PXR ligand. In rifampicin-pretreated PXR-humanized mice, an approximately 60% decrease was observed for both the maximal midazolam serum concentration (C(max)) and the area under the concentration-time curve, as a result of a 3-fold increase in midazolam 1'-hydroxylation. These results illustrate the potential utility of the PXR-humanized mice in the investigation of drug-drug interactions mediated by CYP3A and suggest that the PXR-humanized mouse model would be an appropriate in vivo tool for evaluation of the overall pharmacokinetic consequences of human PXR activation by drugs.

Development of a steroidogenic factor 1/Cre transgenic mouse line

The Cre-loxP strategy provides an approach to disrupt genes in specific tissues and/or cell types, circumventing lethality associated with global knockouts or secondary effects due to gene inactivation at other sites. A critical component is the development of transgenes that target Cre expression to specific cell types. Here, we describe the use of bacterial artificial chromosome (BAC) transgenesis to target Cre expression to tissues that express steroidogenic factor 1 (SF-1, officially designated Nr5a1). Consistent with the SF-1 expression pattern, the SF-1 BAC directed Cre expression to the somatic cells of the gonads, the adrenal cortex, the anterior pituitary, the spleen, and the ventromedial hypothalamic nucleus. This transgene provides a powerful tool to inactivate genes of interest in these tissues.

Targeting iCre expression to murine progesterone receptor cell-lineages using bacterial artificial chromosome transgenesis

Gene-targeting in embryonic stem cells has been the dominant genetic approach when engineering mouse models to query the physiologic importance of the progesterone receptor (PR). Although these models have been instrumental in disclosing the in vivo significance of the progesterone signaling pathway, generation of such mice exacts considerable expenditure of time, effort, and expense. Considering the growing list of new PR mouse models that are urgently required to address the next questions in progestin biology, bacterial artificial chromosome (BAC) recombineering in conjunction with transgenesis was evaluated as an alternative method to accelerate the creation of these models in the future. Using this approach, we describe the generation of three PR-BAC(iCre) transgenic lines in which improved Cre recombinase (iCre) was targeted in-frame, downstream, and under the control of the PR promoter contained within a BAC transgene. Crossing with the ROSA26R revealed that the PR-BAC(iCre) transgenic expresses active iCre only in cell-lineages that express the PR. The specificity of the PR-BAC(iCre) transgene not only underscores the importance of BAC-mediated transgenesis as a quick, easy, and affordable method by which to engineer the next generation of PR mouse models, but also provides a unique opportunity to investigate transcriptional control of PR expression as well as PR structure-function relationships in vivo.

Postembryonic ablation of AgRP neurons in mice leads to a lean, hypophagic phenotype

Agouti-related protein (AgRP) and neuropeptide Y (NPY) are colocalized in arcuate nucleus (arcuate) neurons implicated in the regulation of energy balance. Both AgRP and NPY stimulate food intake when administered into the third ventricle and are up-regulated in states of negative energy balance. However, mice with targeted deletion of either NPY or AgRP or both do not have major alterations in energy homeostasis. Using bacterial artificial chromosome (BAC) transgenesis we have targeted expression of a neurotoxic CAG expanded form of ataxin-3 to AgRP-expressing neurons in the arcuate. This resulted in a 47% loss of AgRP neurons by 16 weeks of age, a significantly reduced body weight, (wild-type mice (WT) 34.7+/-0.7 g vs. transgenic mice (Tg) 28.6+/-0.6 g, P<0.001), and reduced food intake (WT 5.0+/-0.2 vs. Tg 3.6+/-0.1 g per day, P<0.001). Transgenic mice had significantly reduced total body fat, plasma insulin, and increased brown adipose tissue UCP1 expression. Transgenic mice failed to respond to peripherally administered ghrelin but retained sensitivity to PYY 3-36. These data suggest that postembryonic partial loss of AgRP/NPY neurons leads to a lean, hypophagic phenotype.

The best control for the specificity of RNAi

RNA interference (RNAi) is revolutionizing functional genomics. However, there are several reasons to be concerned about the specificity and off-target effects of this technique. A recent paper by Kittler et al. describes a straightforward way to validate RNAi specificity, which exploits the increasing availability of bacterial artificial chromosome (BAC) clone resources. Genetic rescue of the RNAi phenotype by BAC transgenesis is the best control yet described for specificity, and has further implications for reverse genetics.

RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells

RNA interference (RNAi) is a widely used method for analysis of gene function in tissue culture cells. However, to date there has been no reliable method for testing the specificity of any particular RNAi experiment. The ideal experiment is to rescue the phenotype by expression of the target gene in a form refractory to RNAi. The transgene should be expressed at physiological levels and with its different splice variants. Here, we demonstrate that expression of murine bacterial artificial chromosomes in human cells provides a reliable method to create RNAi-resistant transgenes. This strategy should be applicable to all eukaryotes and should therefore be a standard technology for confirming the specificity of RNAi. We show that this technique can be extended to allow the creation of tagged transgenes, expressed at physiological levels, for the further study of gene function.

Microinjection of BAC DNA into the pronuclei of fertilized mouse oocytes.

AbstractMicroinjection of DNA into the pronuclei of fertilized oocytes is one of the two most commonly used methods for gene transfer into the mouse genome (1,2). The first successful attempt to perform this technique was carried out by Lin in 1966 (3), who could show that the early fertilized embryo could survive the mechanical damage of inserting a glass needle into the pronucleus. However, it was not until 1981 that small DNA fragments were integrated into the genome (4). This technique is well described, and has now become a standard procedure (5,6). More recently, the use of larger DNA fragments has been established. Yeast artificial chromosome (YAC) (7,8), P1 artificial chromosome (PAC) (9,10), and bacterial artificial chromosome (BAC) DNA (11,12) can all be used for the generation of transgenic mice. A comprehensive review of BAC and YAC transgenesis is given by Giraldo and Montoliu (13) extensively comparing these applications. Although the basic technique for microinjection of large constructs are similar to those used for shorter DNA segments, there are some special requirements (14). In this chapter, we describe relevant steps for microinjection using large DNA constructs.KeywordsBacterial Artificial ChromosomeEmbryo TransferYeast Artificial ChromosomeInjection NeedleBunsen BurnerThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Expression and Function Profiling of Orphan Nuclear Receptors using Bacterial Artificial Chromosome (BAC) Transgenesis

The long term goal of the Nuclear Receptor Signaling Atlas (NURSA) resides in unraveling the physiological and pathological functions of nuclear receptors (NRs) at the molecular, biochemical and cellular levels. This multi-oriented task requires complementary approaches in order to determine the specific function(s) and precise expression and receptor activity patterns for each individual conventional or orphan receptor. To attain this objective, we have chose to turn to technologies recently made available to engineer bacterial artificial chromosomes (BACs).