WO2006044492A2 - Methods for generating rat embryo-derived cell lines and genetic modification of rat genome - Google Patents

Abstract

The present invention provides novel methods for isolating rat embryo-derived cells and modifying the genome of the rat cells which typically comprise the steps of: constructing a DNA molecule in which desired sequence modifications are contained in a segment of DNA (a 'targeting DNA); introducing the targeting DNA construct into the cell (e.g., by microinjection, electroporation, transfection, or calcium phosphate precipitation); and selecting cells in which the desired sequence modifications have been introduced into the genome via homologous recombination.

Description

Methods for generating rat embryo-derived cell lines and genetic modification of rat genome

FIELD OF THE INVENTION

The present invention relates generally to methods for modifying the genome of rat cells, including rat stem cells and non-stem cells, and more particularly, to methods for generating rat embryo-derived cell lines, modifying a genomic DNA sequence by homologous recombination using rat DNA constructs.

BACKGROUND OF THE INVENTION

( \) Importance of Rat Models

The rat model provides important strengths for the study of human health and disease. The large number of inbred rat models and the vast amount of data (physiological, behavioral, biochemical, cellular, pharmacological, and toxicological, etc.) provide a superb platform on which to build the genetic and genomic tools and resources to delineate the connections between genes and biology. Importantly, in many instances, the rat is the most appropriate experimental model of human disease.

The rat model has made enormous contributions to our present understanding of biological function and behavior. The rat has been a widely studied model system, as demonstrated by the number of publications in the last three decades (nearly 500,000 PubMed publications). Large numbers of rat disease models exist (more than 250 inbred, congenic, mutant, or transgenic rat strains) to explore disease-related variables. The number of transgenic rat models is increasing rapidly. Many of the rat models have already proven their utility for addressing the human condition. Presently rats comprise 28% of all laboratory animals (AALAC) and are highly informative for cardiovascular, pulmonary, renal, endocrinology, reproduction, toxicology, parasitology, immunology, development of dental plaque and gingivitis, polycystic kidney disease, spongioform encephalopathy, alcoholism, nutrition, cancer, growth, diabetes, autoimmune disease, arthritis, asthma, endocrinology, multiple sclerosis, learning, memory, behavior, and neurological health and disease. In some cases, specific aspects of human disease are recapitulated well only in the rat, making these animals a unique resource for studying and identifying genetic pathways relevant human disease. Many examples exist of biological relevance to human health and disease. There are many existing important rat models, just name a few: BB (diabetes), BUF (autoimmune), MNR (emotionality), SHR (hypertension), LOU (plasmacytoma), TMB/TMD (learning), AA/ANA (alcohol avoidance), and RHA, RLA, RCA (avoidance learning). Over 50 years and still up to now, rat models for acute and chronic diseases as well as particular rat strains have been used by the pharmaceutical industry for preclinical drug testing. As a few examples, DA female rats are routinely used for steroid drug therapy trials, since they are debrisoquine poor metabolizers. LEW rats are routinely used for evaluation of anti-inflammatory and immunomodulatory formulations.

There are many more examples available for different aspects, which include:

(a) Hypertension and Heart Disease:

The rat is a model of choice for many physiological studies related to cardiac and vascular function, pulmonary circulation, energetics and metabolism, microcirculation, neural control of cardiovascular, renal and pulmonary function, age and gender related differences, studies of arterial pressure regulation, hypertension, cell and system integrative function, and signal transduction studies. Many inbred rat strains are currently available and well characterized (there are 9 inbred strains for arterial pressure regulation and hypertension alone). The rat hypertension models shared similar physiological markers of the human disorders especially in the models of the spontaneously hypertensive rat, the stroke prone rat, the borderline hypertensive rat and the feminized rat parallel the physiological markers of the human disorders. Rat and mouse have different physiological responses. For example, the mouse is well adapted to dry and hot environment such as desert areas and consequently has a kidney with high capacity to conserve water. This isn't the case for rat and human species. This difference in the conservatory ability lies in the highly activated renin angiotensin system in the digestive system where it regulates the level of water and sodium. This system is very important in rat and human hypertension. As an animal model for heart disease, mouse is very resistant to development heart disease even under high cholesterol and high fat diet. They developed lesions in the root of the aorta. Unlike mouse, rat is very similar to human. The lesions in the rat atherosclerosis model are in the coronary arteries like in humans. Given the same level of cholesterol and triglycerides, the rat atherosclerosis model demonstrates coronary artery disease and decreased survival in contrast to the mouse model.

(b) Neuroscience and drug Addiction:

There is an appreciable depth of knowledge of rat neuroanatomy and neurophysiology. Complex behavioral procedures involved with drug self-administration and developmental studies related to substance abuse, including the behavioral effects of maternal drug exposure, have been extensively characterized in the rat model. Three levels of biological analysis used in the neurological study of alcohol and daig addiction (Intra-Cellular B signal transduction processes; Trans-Cellular B signal transmission processes; Multi-Cellular B signal integration processes) all use the rat successfully to model human biology. Rat studies using models of ethanol self-administration are providing important insight as to how alcohol and aggression interact, with data that appear more related to the human situation than the other models systems. There are several selected alcohol-preferring rat lines that meet the criteria as a model of human alcoholism. Neuroanatomical/stereotaxic injections into the CNS are frequently required to study behaviors. Such techniques are very difficult to carry out on mice and are thus poorly defined. Further more, the size of the rat is necessary to perform important behavior tests associated with many neurological disorders, especially those tests involving site-specific brain cannulas. Behavioral tests relevant to Alzheimer's disease are best developed and validated in rats.

(c) Arthritis and Related Autoimmune Disorders:

Rat models of arthritis and related autoimmune diseases are biologically relevant models to common human diseases such as rheumatoid arthritis, insulin-dependent diabetes, multiple sclerosis, and autoimmune uveitis. More than 200 inbred (e.g., LEW, DA, BB- DP, BB-DR, F344, BN, ACI), congenic (e.g., MHC and other loci), mutant (e.g., athymic nude), or transgenic (e.g., HLA-B27, TNF-alpha, HTLV-I env-pX) rat strains exist in which to explore disease-related variables. Several important models of adjuvant and bacterial cell wall arthritis are only available in the rat, as rats are naturally more susceptible to these disease models. In addition, disease penetrance in mice (as noted in the necessity for repeated injection of potent "adjuvants" for disease induction) is usually less than observed in rats, complicating genetic analyses. Likewise, there are several unique infectious arthritis models available in rat (e.g., Yersinia enterocolitica and Chlamydia trachomatis arthritis). There are unique examples of gene by environment interactions in the rat (induction of insulin-dependent diabetes in BB-DR rats, and induction of arthritis with low potency, non-immunogenic adjuvants in DA rats) as well as responses to therapeutic agents in rat (rats are responsive to non-steroidal anti¬ inflammatory drugs, whereas mice are resistant). Gender-related disease susceptibility profiles in rat are similar to those observed in humans. Female rats are more susceptible (as are humans) to most of the arthritis models than are males. In contrast, male mice are more susceptible than females.

(d) Learning, Memory, and Behavior:

The past 100 years of behavioral research using the rat has revealed the complexity of learning and memory, as well as the multiplicity of brain systems that support it. These studies show that a combination of thorough behavioral characterization and neurobiological investigations can provide major insights into the specific brain systems that mediate memory. Continuing efforts that respect the psychobiological character of rats have recently allowed investigations of even more complex cognitive and memory capacities. For example, the rat's superb learning abilities have been exploited using odors as cues and foraging as a modality for behavioral expression. In this format rats show exceedingly rapid learning of simple discrimination problems - acquiring them typically in 1-2 trials and retaining the information for at least several days. With this capacity in hand, rats have been trained using the same methods on very complex problems such a Piaget's transitive inference task, a test solved by human children at about the age of 7. Rats show robust transitivity and this capacity is fully dependent on the hippocampus.

(e) Endocrinology and Reproductive Biology:

There are various aspects of rat husbandry that provide attractive features for reproductive physiological work; rat pregnancies are more size consistent (compared to the mouse), rat cycling is relatively non-pheromonal (similar to human), rats can be bred quickly after parturition, and rat brains show early sexual dimorphism.

(f) Respiratory and Pulmonary Biology:

Many models of lung disease use rat lungs and/or rat cells. There is a large body of literature in the rat on the neurophysiologic structures, interventions and cardiorespiratory monitoring that enable productive investigation in understanding sleep and breathing. One significant advantage of the rat model is the ability to perform lung function studies. In the rat, sleep, breathing, and cardiac function measurements can be simultaneously recorded. The availability of detailed neuro functional information (in addition to a history of behavior studies) provides an efficient transition from genes to complex behaviors such as sleep. In addition, the rat model mimics many features of human asthma and acute lung injury. Similar phenotypic measures can be accomplished in the rat and human, and have not proven successful in other model systems.

(g) Toxicology and Pharmacology:

Pharmacogenetics is a major emerging research area. Not surprisingly the rat remains a dominant model system for risk assessment of virtually all forms of therapeutics and chemical substances. Insofar as current risk assessment protocols require more than one species it is critical to continue to develop the rat for risk assessment. For example, the acceptance of transgenic animals for risk assessment linked to the increased availability of this technology in rats provides for developing better models systems. Therefore, the combination of classical risk assessment with genetic susceptibility to chemical agents provides unparalleled opportunities for linking the vast databases on drug responses to the genome, as well as increasing our understanding of gene-drug interactions. Cancer. The rat models for breast cancer are good representations of human breast cancer. They are hormonally responsive, can be rapidly induced in virus free animals, and their histopathology and premalignant stages of development resemble those of human breast cancer. The great majority of cancer chemoprevention models in use today are rat based.

(2) The Importance and Usefulness of Rat Stem Cells

Stem cells are important for living organisms for many reasons. In the 3- to 5-day-old embryo, called a blastocyst, stem cells in developing tissues give rise to the multiple specialized cell types that make up the heart, lung, skin, and other tissues. In some adult tissues, such as bone marrow, muscle, and brain, discrete populations of adult stem cells generate replacements for cells that are lost through normal wear and tear, injury, or disease.

Stem cells have two important characteristics that distinguish them from other types of cells. First, they are unspecialized cells that renew themselves for long periods through cell division. The second is that under certain physiologic or experimental conditions, they can be induced to become cells with special functions such as the beating cells of the heart muscle or the insulin-producing cells of the pancreas.

Researchers primarily work with two kinds of stem cells from animals and humans: embryonic stem cells and adult stem cells, which have different functions and characteristics that will be explained in this document. Researchers discovered ways to obtain or derive stem cells from early mouse embryos more than 20 years ago. Many years of detailed study of the biology of mouse stem cells led to the discovery, in 1998, of how to isolate stem cells from human embryos and grow the cells in the laboratory (Science -- Thomson et al. 282 (5391): 1145 : Embryonic Stem Cell Lines Derived from Human Blastocysts). These are called human embryonic stem cells. The embryos used in these studies were created for infertility purposes through in vitro fertilization procedures and when they were no longer needed for that purpose, they were donated for research with the informed consent of the donor. It has been hypothesized by scientists that stem cells may, at some point in the future, become the basis for treating diseases such as Parkinson's disease, diabetes, and heart disease.

Scientists want to study stem cells in the laboratory so they can learn about their essential properties and what makes them different from specialized cell types. As scientists learn more about stem cells, it may become possible to use the cells not just in cell-based therapies, but also for screening new drugs and toxins and understanding birth defects. However, as mentioned above, human embryonic stem cells have only been studied since 1998.

The creation of targeted mutations in the mouse has been a valuable method for the production of animal models of human disease. In addition to the mouse, the rat provides an important model for the study of human health and disease. Detailed information from behavioral studies is more readily available on the rat than the mouse. Additionally, our knowledge of rat physiology is more extensive than that of the mouse, hence the rat system better suited for the study of particular disease states such as certain malignant tumors and cardiovascular disease. Rats are also the model of choice for studies of reproductive physiology, neurobiology, arthritis and the effects of chemoprevention. Furthermore, the larger size of the rat, when compared to the mouse, presents a number of advantages including the availability of more tissue for analysis.

Regenerative medicine requires that stem cells, from whatever source derived, be differentiated (or re-differentiated) into specific body cell types and then physically transplanted into a patient. Differentiation into tissues such as cardiac muscle, blood, and other tissues occurs spontaneously in ES cells being cultured in a dish. Successful application of stem cell technology will require control over the specific kinds of cells into which stem cells differentiate. Control of differentiation and the culture and growth of stem and differentiated cells are important current areas of research for us. Also, some chemicals, such as retinoic acid, can be used to trigger differentiation into specific cell types such as nerve cells.

Among the challenges of medicine, spinal cord injury ranks high. Nerve cells in the spine don't regenerate naturally, and attempts to revive or repair a damaged cord have met with frustration. To bypass this problem, researchers have tried animal experiments replacing ruined nerve cells in animals with transplants of fetal cells. Researchers at Washington University School of Medicine in St. Louis reported in 2000 that they have restored leg movement in injured rats by transplanting cells into the injury site 9 days after the rats received a crushing blow to the spine. The scientists used mouse-embryo stem cells modified to ensure they would grow into basic nerve cells and associated cells.

Memory in aged rats has been greatly improved by transplanting human neural stem cells, taking scientists a step closer to a cure for Alzheimer's and Parkinson's.

In 2001, Researchers from the University of Illinois in Chicago found aged rats that had received the transplants were able to perform as well as younger rats without impaired memory.

Recently, researchers from Kyoto University have now shown that dopamine-producing neurons (DA neurons) generated from monkey embryonic stem cells and transplanted into areas of the brain where these neurons have degenerated in a monkey model of Parkinson disease, can reverse parkinsonism (2005).

Rat embryonic stem cells will be very useful to help scientists understand the basis for treating diseases such as Parkinson's disease, diabetes, and heart disease. All above experiments could be effectively and economically done with rat embryonic stem cells and/or rat models. The larger size of the rat, when compared to the mouse, presents a number of advantages including the availability of more tissue for analysis.

In recent years, several attempts were made to establish pluripotent rat ES cells, but the cells could be maintained in culture for only a very short time (Takahama et al. 1998; Ouhibi et al. 1995 ; Stranzinger 1996), or the experiments could not be repeated or contaminated with mouse embryonic stem cells (Iannaccone et al. 1994; Brenin et al. 1997). The pharmaceutical industry is concentrating its research effort on the development speed of new drugs. In order to create innovative drugs and treat complex pathologies, the industry needs effective biological models (cell and animal) offering high predictability for human responses to candidate drugs.

In neuroscience, metabolic disorders, cardiovascular diseases, or toxicology, the rat is an animal that has a very similar physiology to that of humans. This makes it a first-choice model for the pharmaceutical industry. So far, only the mouse genome allows targeted gene modification.

The rat is a reference animal model for physiological studies and for the analysis of multigenic human diseases such as hypertension, diabetes, and neurological disorders. Genetic manipulation in the rat is hampered by the lack of suitable technologies such as embryonic stem cells (ES), which are routinely used to generate targeted mutations in the mouse. Cloning through somatic cell nuclear transfer (SCNT) is a potential alternative approach in species for which ES technologies are unavailable. In 2003, genOway (Lyon, France), Europe's leading provider of transgenic cellular and animal models, and its partner INRA (Institut National de Ia Recherche Agronomique), announce that they have succeeded in producing the world's first cloned rats with primary rat embryonic fibroblasts (Zhou et al.2003).

Embryonic stem cells could be much better than somatic cells on increasing the success rate. A successful example is that the frequency of term pregnancies for blastocysts derived from ES-I ike cells was higher than those of early pregnancies and maintained pregnancies after nuclear transplantation (NT) with bovine somatic cells (Saito et al. 2003).

(3) Importance and Usefulness of Targeted Rat Stem Cell Lines

The targeted rat stem cells can be used in many areas, as previously shown in the mouse. By using gene targeting technique, nearly 1000 knockout mice have been produced over the last two decades. Generating knockout mice, however, is a time-consuming procedure. Also, an unexpected embryonic lethality sometimes prevents us from examining the function of the gene in specific tissues. The homozygous knockout ES cells have been shown useful to determine the role of the genes in the mediation of various cellular activities such as proliferation, differentiation, apoptosis, survival, transformation, and so on. Furthermore, with the recent advance of in vitro differentiation techniques, it is now feasible to rapidly determine the role of specific molecules in particular tissues.

In vitro differentiation experiments using ES cells bearing homozygous null-mutations of particular genes have been reported to be particularly useful to analyze the contribution of a gene product during embryonic development. Indeed, when investigated, mutation defects in animals paralleled modifications observed in ES in vitro differentiation systems. Similar defects in vascular development were detected both in vivo and in vitro after VEGF gene inactivation.

The rat stem cell lines can be targeted and used in many areas. There are a large amount of experimental procedure and data from targeted mouse stem cells. Based on the difference between mouse and rat, the results from rat may provide more useful information than from mouse.

(a) Immunological and Inflammatory Diseases

As for a example, we may target the MEKK 1/2 genes in the rat stem cells and it is possible to study the function of the gene in targeted rat stem cells, as shown in the mouse MEKK2^7" or MEKKl^"7" stem cell - derived mast cells (ESMC) studies (Garrington at al. 2000). As we already known, Mast cells are centrally important in inflammatory and immediate allergic reactions (Metcalfe et al., 1997). MAPKs are regulated by a family of proteins known as MAPK kinases (MKKs), which are in turn regulated by a family of MKK kinases (MKKKs or MAP3Ks) (Widmann et al., 1999).

MEKK2 is a 70 kDa member of the MEKK group of MAP3Ks that has been shown to regulate the JNK and ERK5 pathways (Blank et al., 1996; Sun et al., 2000). It has shown recently that MEKK2 is recaiited to the T-cell receptor (TCR) signaling complex upon presentation of antigen to T cells (Schaefer et al., 1999). MEKK2 was activated in response to antigen presentation and was required for stabilization of conjugates of T cells and antigen-presenting cells. MEKKl and MEKK3 were not recruited to the TCR signaling complex in response to antigen, showing the selectivity of this response to MEKK2. Also, MEKK2^~A embryonic stem (ES) cells were used for Rag2^"A blastocyst complementation to define the role of MEKK2 in lymphocyte development. It was found that MEKK2 was required for B- and T⁺-CeIl development beyond the pre-BCR and pre- TCR signaling checkpoints, respectively. Targeted disruption of the MEKK2 or MEKKl gene was used to abolish expression of the respective kinases in ESMC. Transcription of specific cytokines in response to IgE or c-Kit ligand was markedly reduced in MEKK2^"Λ ESMC relative to wild-type ESMC. Cytokine production in MEKKl^7" ESMC was similar to that of wild-type ESMC, demonstrating the specificity of MEKK2 in signaling cytokine gene regulation. MEKK2^"Λ ESMC also lost receptor-mediated stimulation of JNK. In contrast, JNK activation in response to UV irradiation was normal, showing that MEKK2 is required for receptor signaling but not for cellular stress responses. MEKK2 is the first MAP3K shown to be required for mast cell tyrosine kinase receptor signaling controlling cytokine gene expression.

(b) Obesity and Diabetes

As for another example, we may target the transcription factor peroxisome proliferator- activated receptor γ (PP ARγ) in the rat stem cells and it is possible to study the function of the gene in targeted rat stem cells, as shown the mouse PPARγ ^"Λ stem cell studies. As we already known, PP ARγ has been associated with several distinct biological programs. It was cloned independently as a new member of the PPAR subgroup of nuclear hormone receptors and as a transcriptional regulator of fat-specific gene expression. Subsequently, it has become clear that PP ARγ is the functional receptor for an interesting class of insulin-sensitizing drugs called thiazolidinediones (TZDs), which are currently used in the treatment of type 2 diabetes mellitus. PP ARγ has also been recently implicated in the differentiation of other cells and tissues, such as macrophages, breast, and colon, and mutations of PP ARγ that destroy receptor function have been found in sporadic human colon cancer. PPARγ null cells showed little or no contribution of null cells to adipose tissue, whereas most other organs examined do not require PPARγ for proper development. In vitro, the differentiation of ES cells into fat is shown to be dependent on PPARγ gene dosage. These data provide direct evidence that PPARγ is essential for the formation of fat (Rosen et al. 2002).

(c) Muscular Dystrophy

As for another example, we may target the laminin α2 chain gene in the rat stem cells and it is possible to study the function of the gene in targeted rat stem cells, as shown in the mouse laminin <x2 chain gene ^7" stem cell studies. As we already known, mutations in the gene coding for the α2 chain of laminin-2 and -4 (merosin) cause a severe form of congenital muscular dystrophy in humans and mice. Mutations in the gene coding for the α2 chain of laminin-2 and -4 (merosin) cause a severe form of congenital muscular dystrophy in humans and mice. To establish a defined model for in vitro and in vivo studies of the role of laminin α2/merosin in development and cell and tissue function, it has showed that mouse laminin α2 chain gene ^~'^~ stem cell differentiate normally in vitro , giving rise to cardiomyocytes, myotubes, and smooth muscle cells in addition to many other cell types. However, the myotubes that are formed are unstable. They detach, collapse, and degenerate, a process which is initiated at the appearance of the mature, contractile phenotype of the cells. It lead to the conclusion that the detachment and death of contracting myotubes in vitro has its counterpart in vivo and that contraction-induced myofiber damage, along with the lack of survival cues provided by laminin α2/merosin, is a significant contribution to muscle degeneration in merosin-deficient muscular dystrophy (Kuang et al. 2002).

(d) Vascular Disease

As for another example, we may target the Vascular endothelial-cadherin (VE-cadherin) gene in the rat stem cells and it is possible to study the function of the gene in targeted rat stem cells, as shown in the mouse VE-cadherin ^"Λ stem cell studies. As we already known, vasculogenesis is a process whereby angioblasts differentiate in situ to endothelial cells that connect and form primitive blood vessels. Vascular endothelial-cadherin (VE- cadherin) is exclusively expressed in endothelial cells and is strictly located at cell-to-cell junctions. As the other members of the cadherin family, VE-cadherin is able to mediate a homotypic type of cellular interaction in a Ca ⁺-dependent manner. In the mouse embryo, VE-cadherin transcripts are detected at the earliest stages of vascular development. To ascertain if VE-cadherin expression is required for the assembly of endothelial cells into vascular structures, VE-cadherin mouse ES cells was generated by gene targeting and examined the consequences on vascular development of ES-derived embryoid bodies (EBs). In contrast to wild-type EBs, endothelial cells remained dispersed and failed to organize a vessel-like pattern in VE-cadherin ES-derived EBs. However, dispersed VE-cadherin^ ^~ ES-derived endothelial cells expressed a large range of other endothelial markers. Moreover, the targeted null-mutation in the VE-cadherin locus did not interfere with the hematopoietic differentiation potential of ES cells. These in vitro experiments are consistent with a pivotal role of VE-cadherin in vascular structure assembly (Vittet at al. 1997).

(e) Reporter Gene system

As for an example, we may setup a Cell-based drug screens for regulators of gene expression. This assay may involve (a) contacting a mammalian cell comprising a knock-in mutant of a targeted native allele encoding a reporter of gene expression, wherein the expression of the reporter is under the control of the gene expression regulatory sequences of the native allele, with a candidate agent under conditions whereby but for the presence of the agent, the reporter is expressed at a first expression level; and, (b) measuring the expression of the reporter to obtain a second expression level, wherein a difference between the first and second expression levels indicates that the candidate agent modulates gene expression. As for more detail, This assay can be used for screening for agents which regulate the level of targeted gene expression in a natural context. Such agents find use in modulating a wide variety of physiological manifestations of gene expression.

The subject assays are cell-based and generally involve contacting a mammalian cell comprising a mutant of a native allele encoding a reporter of the targeted gene expression, wherein the expression of the reporter is under the control of the native gene expression regulatory sequences of the native targeted allele, with a candidate agent under conditions whereby but for the presence of the agent, the reporter is expressed at a first expression level; and, measuring the expression of the reporter to obtain a second expression level, wherein a difference between the first and second expression levels indicates that the candidate agent modulates the expression of the targeted gene.

The mutant generally results from replacement of a portion of the native allele with a sequence encoding the reporter. For example, the cell may be a progeny of, a clone or, or genetically identical to a genetic knock- in cell made by homologous recombination of the native allele with a transgene comprising a sequence encoding the reporter flanked by flanking sequences capable of effecting the homologous recombination of the transgene with the native allele, a positive selectable marker positioned inside the flanking sequences and optionally, a negative selectable marker positioned outside the flanking sequences.

Preferred reporter genes can be readily expressed by the rat stem cells and provide products that are readily detected and quantified. Exemplary reporter genes include .beta.-galactosidase, CAT, GFP, luciferase, and bacteria NTR. The mutated locus may also comprise a positive selection marker such as an antibiotic resistance gene, e.g. neomycin, residual from the initial construction of the mutation. Alternatively, such residual sequences may be lost or removed, e.g. using a Loxp-CRE recombination system, in the course of cell passage or animal reproduction.

In summary, the technology of gene targeting through homologous recombination has been extremely useful for elucidating gene functions in mice. It has become routine in many laboratories around the world to produce mice with specific genetic modifications including gene disruption, gene replacement, and even engineered chromosomal translocation. However, it has been extremely difficult to alter genes in mammals, other than the mouse, by homologous recombination. There is no report of gene targeting with rat cells. Targeted gene disruption by homologous recombination in rats will be very useful to help scientists understand the basis for treating diseases such as Parkinson's disease, diabetes, and heart disease.

SUMMARY OF THE INVENTION

The present invention overcomes the problems in generating transgenic rats described in the art by providing methods for the isolation of rat stem cell lines, methods for transforming both the rat embryonic stem cells and the cultured cell lines, and using these transformed cells and cell lines to generate transgenic rats. The efficiency at which transgenic rats are generated by the present invention is greatly increased, thereby allowing the use of homologous recombination in producing transgenic rats.

Accordingly, the present invention provides a method of isolating rat embryonic stem cells on feeder cells, the feeder cells at a density of between about 50 cells/cm², in a culture medium comprising an effective amount of neonatal rat liver conditional medium and neonatal rat serum.

In another aspect, the invention provides a method of growing rat embryonic stem cells from a rat species, comprising plating a composition comprising rat embryonic stem cells from an embryo of said rat species on feeder cells, in a culture medium including exogenously added leukemia inhibitory factor.

In particular aspects of the present invention, the rat embryonic stem cells comprise at least a first exogenous DNA segment. Rat embryonic stem cells comprising exogenous DNA are referred to as genetically transformed rat embryonic stem cells. In further embodiments, the rat embryonic stem cells are provided with an exogenous, selected DNA segment by electroporation, particle bombardment or calcium phosphate precipitation. In certain aspects of the invention the composition comprising rat embryonic stem cells is provided with a selected DNA segment and the rat embryonic stem cells that contain the selected DNA segment are selected and optionally separated away from the rat embryonic stem cells of the composition that do not contain the selected DNA segment. The isolated composition comprising the rat embryonic stem cells is grown on a layer of feeder cells. The feeder cells provide a microenvironment conducive to the growth of the rat embryonic stem cells. The feeder cells provide growth factors to the growing rat embryonic stem cells, as well as providing an extracellular matrix. In certain aspects of the present invention, the feeder cell lines may be engineered to express selected growth factors. Thus in certain embodiments of the present invention, the feeder cells may comprise at least a exogenous DNA sequence.

The feeder cells are inactivated prior to use, preferably by gamma-irradiation or using mitomycin C. In preferred embodiments of the present invention, the feeder cells are inactivated with cobalt radiation or cesium radiation.

The present invention also provides methods for culturing the isolated rat embryonic stem cells in an appropriate medium. As discussed above, the feeder cells, neonatal rat liver conditional medium and neonatal rat serum provide growth factors to the growing rat embryonic stem cells, however, the amount of endogenous growth factors provided may vary from preparation. Therefore, in certain aspects of the invention exogenously added growth factors may be added to supplement the endogenous supply.

A growth factor that is critical for growth of the rat embryonic stem cells of the present invention is bone morphogenetic protein 4 (BMP4). As is the case with each of the growth factors described herein, bone morphogenetic protein 4 can be utilized from a variety of mammalian sources, including, but not limited to, porcine, bovine, ovine, caprine, equine, murine.

Other growth factors may be added to the medium in an amount effective to improve the growth characteristics of the rat embryonic stem cells, or to help maintain the rat embryonic stem cells in an undifferentiated state. Thus, in particular embodiments, the culture medium may also comprise an effective amount of leukemia inhibitory factor. In preferred embodiments of the present invention, the culture medium may also comprise an effective amount of L-glutamine. In particular aspects, the culture medium comprises L-glutamine at a concentration of between about 0.1 mM and about 50 mM. In more preferred embodiments, the culture medium comprises L-glutamine at a concentration of between about 1 mM and about 20 mM.

In certain embodiments, the culture medium may also comprise an effective amount of Dulbecco's modified Eagle's media. The Dulbecco's modified Eagle's media may be either low sodium Dulbecco's modified Eagle's media or high sodium Dulbecco's modified Eagle's media.

Culture media comprising combinations of different growth factors are also contemplated for use in the present invention. Thus, in certain aspects of the present invention, the culture medium comprises an effective amount of human bone morphogenetic protein 4 and an effective amount of at least one of uteroferrin, .alpha.2-macroglobulin, leukemia inhibitory factor, soluble stem cell factor, amino acids non-essential to said rat, L- glutamine, .beta.-mercaptoethanol, Dulbecco's modified Eagle's media or Ham's FlO media. In further aspects, the culture medium comprises an effective amount of human bone morphogenetic protein 4 and a combined effective amount of at least two of uteroferrin, .alpha.2-macroglobulin, leukemia inhibitory factor, soluble stem cell factor, amino acids non-essential to said rat, L-glutamine, .beta.-mercaptoethanol, Dulbecco's modified Eagle's media or Ham's FlO media.

The invention also provides methods wherein the plated rat embryonic stem cells are maintained in an undifferentiated state for about 2 passages, about 3 passages, about 4 passages, about 5 passages, about 6 passages, about 7 passages, about 8 passages, about 9 passages, about 10 passages, about 11 passages, about 12 passages, about 13 passages or about 14 passages. In other embodiments of the present invention, the plated rat stem cells are maintained in an undifferentiated state for about 20 passages, about 30 passages, about 50 passages or about 100 passages. Additionally, the present invention provides a method of preparing rat embryonic stem cells that contain a selected DNA segment, that may comprise introducing a selected DNA segment into a composition comprising rat embryonic stem cells to obtain candidate rat embryonic stem cells that contain the selected DNA segment.

In exemplary methods of the present invention, the selected DNA segment is introduced into the rat stem cell by electroporation. In other methods, the selected DNA segment is introduced into the rat stem cell by particle bombardment, calcium phosphate transformation or by viral transformation.

In certain embodiments, the selected DNA segment may comprise at least a first coding region encoding a selected protein, wherein the coding region is expressed in one or more of the rat embryonic stem cells. In further embodiments, the first coding region encodes a selected disease resistance, carcass composition, weight gain, coat composition or milk component protein. In other embodiments, the first coding region encodes a selected marker protein. In exemplary embodiments, the first coding region encodes green fluorescent protein that has been adapted to increase expression in the rat species. A protein is "adapted to increase expression in" a rat species by altering the coding sequence of the protein to use codons that are preferred for use in the particular rat species desired for use. In still other embodiments, the first coding region encodes a neomycin resistance protein. In further embodiments, the first coding region encodes GP63, myelin basic protein, hCD59, Factor IX, .alpha. -antitrypsin, .alpha. -casein, an interleukin or Bcl-2.

In exemplary embodiments of the present invention, the selected DNA segment may also comprises a second coding region encoding a selected protein. In particular embodiments of the present invention, the first coding region may encodes a selected non-marker protein and the second coding region encodes a selected marker protein.

In embodiments wherein expression of the selected DNA segment is desired, the DNA segment is operatively positioned under the control of a promoter, exemplified by, but not limited to, the CMV promoter, the Oct-4 promoter or the pgk promoter, that expresses the DNA segment in the rat embryonic stem cells. In other embodiments of the present invention, the selected DNA segment is operatively positioned in reverse orientation under the control of the promoter, wherein the promoter directs the expression of an antisense product.

In still other embodiments of the instant invention, the DNA segment comprises two selected DNA regions that flank the coding region, thereby directing the homologous recombination of the coding region into the genomic DNA of a rat species. In more preferred embodiments, the selected DNA regions correspond to distinct sequences in the genomic DNA of the rat species. In exemplary embodiments, the isolated DNA regions correspond to the Oct-4 gene, or regions that flank the Oct-4 gene.

In still other embodiments of the present invention, the DNA segment comprises two selected DNA sequences that flank the DNA segment and allow for excision of the DNA segment under appropriate conditions. In particularly preferred embodiments, the DNA sequences are loxP sites.

In particular embodiments of the present invention, the transgenic rat can be generated by a method comprising injecting the rat embryonic stem cells that contain said selected DNA segment into a blastocyst from said rat species. In certain aspects, the transgenic rat can be generated by a method comprising injecting the rat stem that contain the selected DNA segment into a blastocyst from the rat species, transferring the blastocyst into a synchronized recipient female of rats to produce a pregnant rat, and allowing gestation in the pregnant rat to proceed for a period of time sufficient to allow the development of a viable transgenic rat. In further embodiments, the viable transgenic rat is obtained by natural birth, while in other embodiments, the viable transgenic rat can be obtained by surgically removing the viable transgenic rat from the recipient female.

In other aspects of the present invention, the transgenic rat can be generated by a method comprising isolating a nucleus from the rat embryonic stem cells that contain the selected DNA segment and injecting the nucleus into an enucleated oocyte from the rat. In particular embodiments, the transgenic rat can be generated by a method comprising, isolating a nucleus from the rat embryonic stem cells that contain the selected DNA segment and injecting the nucleus into an enucleated oocyte from said rat, transferring the oocyte into a synchronized recipient female of the rat to produce a pregnant rat, and allowing gestation in the pregnant rat to proceed for a period of time sufficient to allow the development of a viable transgenic rat.

In still other embodiments of the present invention, the transgenic rat can be generated by a method comprising aggregating the rat embryonic stem cells of the rat species that contain the selected DNA segment with an early stage embryo of the rat species. In certain aspects, the transgenic rat can be generated by a method comprising aggregating the rat embryonic stem cells of the rat species that contain the selected DNA segment with an early stage embryo of the rat species, transferring the embryo into a synchronized recipient female of the rat species to produce a pregnant rat, and allowing gestation in the pregnant rat to proceed for a period of time sufficient to allow the development of a viable transgenic rat.

The present invention provides novel methods for modifying the genome of a mammalian cell, specifically, rat cell, comprising the steps of: constructing a DNA molecule in which desired sequence modifications are contained in a segment of DNA (a "targeting DNA"), introducing the targeting DNA construct into the cell (e.g., by microinjection, electroporation, transfection, or calcium phosphate precipitation); and selecting cells in which the desired sequence modifications have been introduced into the genome via homologous recombination.

Although the present invention has been applied to laboratory rat strains such as Brown Norway, Fisher 344 and hybrid (Brown Norway x Fisher 344), the invention will be also useful for gene targeting in other rat strains. The typical rat strains used in laboratories tend to be fairly inbred and, as a result, there is smaller likelihood of sequence divergence in an allele derived from different lines (see, e.g., Bishop, C, et al., Nature 315:70-72 (1985)). A preferred cell type for targeting the genome of a mammalian organism is the embryonic stem cell. Preferably, the DNA construct contains an antibiotic resistance marker and the cells are first selected on a medium containing the antibiotic.

The present invention also provides novel methods for creating genetically modified animals comprising the steps of: modifying the genome of embryonic stem cells derived from the animal, as described above; introducing the modified embryonic stem cells into blastocysts derived from the same species of animal; and using a pseudo- pregnant female to carry the chimeric animal to term. The resulting chimeric animal can in turn be bred to obtain non-chimeric animals in which the desired genetic alteration has been stably inherited through germ-line transmission. Specifically, in the present invention the gene for apolipoprotein II (All) is modified through the above steps to produce non-chimeric rats with modified All gene. Since All plays an important role in the metabolism of high density lipoprotein (HDL), a good cholesterol, this rat knockout rat provides an important model to study atherosclerosis, myocardial infarction and stroke. The animals can be used to screen for drugs that are effective as therapeutics or diagnostics of heart disease.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. shows rat blastocysts of 4.5 dpc derived from Brown Norway or Fisher 344 rats.

FIG. 2. shows a typical ES colony of rat ES cell lines derived from Brown Norway rats or

Fisher 344 rats.

FIG. 3 shows the embryoid body of WH7 cell line.

FIG. 4 shows the rat ES genomic DNA of apolipoprotein All, cut by Nsil, where the wildtype is 8.6 KB and the knockout is 7.5 KB

FIG.5. shows another embodiment of the rat ES genomic DNA of apolipoprotein All, cut by Nsil, where the wildtype is 14 KB and the knockout is 11 KB. DETAILED DESCRIPTION OF THE INVENTION

1. Generation of Rat Stem Cell Lines

Embryonic stem cells isolated from the inner cell mass of the preimplantation embryo possess the ability to proliferate indefinitely in an undifferentiated state, are capable of differentiating in vitro and in vivo, and can contribute to the formation of normal tissues and organs of a chimeric individual when injected into a host embryo. Differentiation can be stimulated in vitro by modifying culture conditions, and in vivo by injection of ES cells into athymic mice. When allowed to differentiate in vitro, ES cells form structures known as embryoid bodies, which closely resemble the embryo-proper portion of the 5- day mouse embryo.

The ability to colonize the germ line following culture and genetic manipulation has made ES cells a powerful tool for the modification of the genome in the mouse species. Chimeras produced between genetically modified ES cells and normal embryos have been used to study in vivo gene regulation as well as germ-line transmission of introduced genes. In addition, ES cells have been used to study targeted modification of genes by homologous recombination.

Although the majority of the research on ES cells has been done in the mouse, attempts at developing the ES technology in other mammalian species have been reported by several investigators. Doetschman et al. (1988), showed that ES cells can be isolated from hamster embryos using feeders composed of murine primary embryonic fibroblasts. Several investigators using STO as feeder layers, have reported the isolation of porcine embryo-derived cell lines with ES-like morphology and a limited ability to differentiate in vitro and in vivo.

Rat embryonic stem cells will be very useful to help scientists understand the basis for treating diseases such as Parkinson's disease, diabetes, and heart disease. The larger size of the rat, when compared to the mouse, presents a number of advantages including the availability of more tissue for analysis. In recent years, several attempts were made to establish pluripotent rat ES cells, but the cells could be maintained in culture for only a very short time (Takahama et al. 1998; Ouhibi et al. 1995; Stranzinger 1996), or the experiments could not be repeated or contaminated with mouse embryonic stem cells (Iannaccone et al. 1994; Brenin et al. 1997).

(1) Embryo Isolation

Embryos are collected from pregnant female rats of the selected rat strains. After the rats are sacrificed, and the uterus is removed. Embryos were flushed from the. uterus of rats with FHM medium (Specialty Media) on the fifth day after natural mating (4.5 dpc). Well-developed blastocysts were transferred to ES medium in a 96-well plate with γ-irradiated mouse embryonic fibroblasts as feeders. ES cell medium contains 15v/v FBS (HyClone), 1 mM non-essential amino acids (GIBCO), 0.1 mM 2-mercaptoethanol (GIBCO), 10 ng/ml human 1% Glutamine, 1 % pen/strep, 1000 units/mL Leukemia-inhibiting factor (LlF) (Chemicon: ESGl 107), 15 ng/ml human bone morphogenetic protein 4 (BMP4), 5% v/v neonatal rat liver conditional medium, 1% v/v neonatal rat serum.

(2). Isolation and Culturing of Rat Embryonic Stem (ES) Cells

Embryos were flushed from the uterus of Brown Norway, Fisher344 and hybrid (Brown Norway X Fisher 344 females with FHM medium (Specialty Media) on the fifth day after natural mating (5 dpc). Well-developed blastocysts were transferred to ES medium in a 96-well plate with γ- irradiated mouse embryonic fibroblasts as feeders. ES cell medium contains 15v/v FBS (HyClone), 1 mM non-essential amino acids (GIBCO), 0.1 mM 2-mercaptoethanol (GIBCO), 1% Glutamine, 1 % pen/strep, 1000 units/mL Leukemia-inhibiting factor (LIF) (Chemicon: ESGl 107), 15 ng/ml human bone morphogenetic protein 4 (BMP4), 5% v/v neonatal rat liver conditional medium, 1% v/v neonatal rat serum. After 3-6 days, the central mass of each explant was trpsinized with 0.25% Trypsin/EDTA for 5 min. Resultant primary ES cell colonies were individually passaged into wells of a 96 well plate with feeders. Thereafter, cells were expanded by trypsinization of the entire culture. (a) Feeder Cells

The isolated rat ES cells are grown on a layer of feeder cells. Types of feeder cells that may be used in the present invention are embryonic fibroblasts from mouse and preferably from rat. STO cells can also be used (mouse embryonic fibroblast cells; Ware and Axelrad, 1972). The feeder cells provide growth factors to the growing rat ES cells, but the amount of endogenous growth factors provided is variable from preparation to preparation. Therefore, exogenously added growth factors may be added to supplement the endogenous supply. Additionally, in particular aspects of the invention, the inventors contemplate engineering feeder cell lines to express selected growth factors, for example membrane-associated stem cell factor and basic fibroblast growth factor.

The feeder cells are inactivated prior to use, preferably by gama-irradiation with agents such as cobalt or cesium, or using mitomycin C. The inactivated feeder cells are allowed to culture prior to use in culturing rat ES cells, preferably for 1-3 hours, but longer and shorter culture times are possible.

(b) Media Composition

The present invention provides compositions for rat ES cell growth media. The rat ES cell can be grown on inactivated feeder cells in media directly after isolation, upon thawing from cryopreservation, or after transformation. Preferred media for use in the present invention is high glucose Dulbecco's modified Eagle's media. Preferably, the media is supplemented with L-glutamine. Additional preferred media is supplemented with .beta.-mercaptoethanol, and still other preferred media is supplemented with 100 nM of non-essential amino acids (GIBCO). More preferred for use in the present invention is fully supplemented media, additionally comprising one or more of the following growth factors.

a. human bone morphogenetic protein 4 (BMP4)

b. neonatal rat liver conditional medium c. neonatal rat serum.

d. LIF

(c) Culture conditions

Optimization of conditions such as pH, percent CO. sub.2, pθ.sub.2 and temperature for maximum growth of primordial germ cell cultures are well known to those of skill in the art. The preferred embryo-derived stem cell culture conditions are about 5% - 10%CO.sub.2 at about 38. degree. C. in a humidified atmosphere.

2. Gene Targeting in Rat Stem Cell Lines

Gene targeting now provides the means for creating new strains of mice with mutations in virtually any gene. In accordance with the present invention, gene targeting can be used to modify the genome of rat stem cells besides mouse cells, using an efficient technique involving homologous recombination between exogenous "targeting DNA" and rat genome. By introducing an exogenous "targeting DNA" into eukaryotic cells, selecting for cells in which the targeting DNA has been stably integrated into the recipient cell genome is readily accomplished.

There are two general events believed to be responsible for stable integration. In homologous recombination, the incoming DNA interacts with and integrates into a site in the genome that contains homologous DNA sequence. In non-homologous ("random" or "illicit") integration, the incoming DNA is not found at a homologous sequence in the genome but integrates randomly. In general, studies with higher eukaryotic cells have revealed that the frequency of homologous recombination is far less than the frequency of random integration. The ratio of these frequencies has direct implications for "gene targeting" which depends on integration via homologous recombination (i.e. recombination between the exogenous "targeting DNA" and the corresponding "target DNA" in the genome).

The extension of gene targeting to other mammalian species will provide an additional boost to the study of mammalian biology. Gene targeting represents a major advance in the ability to selectively manipulate animal cell genomes. Using this technique, a particular DNA sequence can be targeted and modified in a site-specific and precise manner. Different types of DNA sequences can be targeted for modification, including regulatory regions, coding regions and regions of DNA between genes. Examples of regulatory regions include: promoter regions, enhancer regions, terminator regions and introns. By modifying these regulatory regions, the timing and level of expression of a gene can be altered. Coding regions can be modified to alter, enhance or eliminate, for example, the specificity of an antigen or antibody, the activity of an enzyme, the composition of a food protein, the sensitivity of protein to inactivation, the secretion of a protein, or the routing of a protein within a cell. Introns and exons, as well as inter-genic regions, are suitable targets for modification.

Modifications of DNA sequences can be of several types, including insertions, deletions, substitutions, or any combination of the preceding. A specific example of a modification is the inactivation of a gene by site-specific integration of a nucleotide sequence that disrupts expression of the gene product. Using such a technique to "knock out" a gene by targeting will avoid problems associated with the use of antisense RNA to disrupt functional expression of a gene product. For example, one approach to disrupting a target gene using the present invention would be to insert a selectable marker into the targeting DNA such that homologous recombination between the targeting DNA and the target DNA will result in insertion of the selectable marker into the coding region of the target gene.

It may be preferable to incorporate a selectable marker into the targeting DNA which allows for selection of targeted cells that have stably incorporated the targeting DNA. This is especially useful when employing relatively low efficiency transformation techniques such as electroporation, calcium phosphate precipitation and liposome fusion, as discussed below, where typically fewer than 1 in 1000 cells will have stably incorporated the exogenous DNA.

Examples of selectable markers include: genes conferring resistance to compounds such as antibiotics, genes conferring the ability to grow on selected substrates, genes encoding proteins that produce detectable signals such as luminescence. A wide variety of such markers are known and available, including, for example, antibiotic resistance genes such as the neomycin resistance gene (neo). Selectable markers also include genes conferring the ability to grow on certain media substrates such as the tk gene (thymidine kinase) or the hprt gene (hypoxanthine phosphoribosyltransferase) which confer the ability to grow on HAT medium (hypoxanthine, aminopterin and thymidine); and the bacterial gpt gene (guanine/xanthine phosphoribosyltransferase) which allows growth on MAX medium (mycophenolic acid, adenine, and xanthine). See Song, K-Y., et al. Proc. Nat'l Acad. Sci. U.S.A. 84:6820-6824 (1987). Other selectable markers for use in mammalian cells, and plasmids carrying a variety of selectable markers, are described in Sambrook, J., et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1989).

If a selectable marker is used, the preferred location of the marker gene in the targeting construct will depend on the aim of the gene targeting. For example, if the aim is to disrupt target gene expression, then the selectable marker can be cloned into targeting DNA corresponding to coding sequence in the target DNA. Alternatively, if the aim is to express an altered product from the target gene, such as a protein with an amino acid substitution, then the coding sequence can be modified to code for the substitution, and the selectable marker can be placed outside of the coding region, in a nearby intron for example.

If the selectable markers will depend on their own promoters for expression and the marker gene is derived from a very different organism than the organism being targeted (e.g. prokaryotic marker genes used in targeting mammalian cells), it is preferable to replace the original promoter with transcriptional machinery known to function in the recipient cells. A large number of transcriptional initiation regions are available for such purposes including, for example, metallothionein promoters, thymidine kinase promoters, beta-actin promoters, immunoglobulin promoters, SV40 promoters and human cytomegalovirus promoters. A widely used example is the pSV2-neo plasmid which has the bacterial neomycin phosphotransferase gene under control of the SV40 early promoter and confers in mammalian cells resistance to G418 (an antibiotic related to neomycin). A number of other variations may be employed to enhance expression of the selectable markers in animal cells, such as the addition of a poly(A) sequence and the addition of synthetic translation initiation sequences. Both constitutive and inducible promoters may be used.

In some cases, it may be desirable for the modification sequences (including selectable markers) to alter the transcriptional activity of the target gene. However, if selectable markers are used and it is not desirable to affect transcriptional activity of the target gene, it will be preferable to use selectable markers with an inducible promoter and/or to include a transcription termination sequence downstream of the selectable marker. A variety of inducible promoters and transcription termination sequences are known and available. See, e.g., Sambrook, supra.

Where the target gene is highly expressed or readily inducible, it may be advantageous to use selectable markers lacking their own promoters as a way to further enhance the frequency of obtaining homologous recombinants. In that way, the likelihood of the selectable marker being highly expressed upon integration into the genome will be much greater for homologous recombination events (where the promoterless gene will have been placed in the vicinity of the target gene promoter) than for random integration into the genome.

Target genes can also be modified by deletions. In the case of a deletion, the sequence to be deleted will be absent or removed from the corresponding targeting DNA and thus the "modification sequence" will constitute a missing sequence relative to the target DNA. The deletion will generally cover a portion of one or more exons and may include introns and flanking non-coding regions such as regulatory regions. The deletion may be as small as one base pair or as large as tens of thousands of base pairs.

Another specific form of modification is the introduction of a new gene into the animal cell genome. By flanking the new gene with sequences substantially isogenic with target DNA in the host cell, it is possible to introduce the gene in a site-specific fashion at the targeted location. Using this approach, a gene from any source (e.g., bacterial, plant, animal) can be introduced into an animal cell to impart new characteristics to the cell or to allow the animal cell to produce desired polypeptides which can then be isolated from the animal or from its cells in vitro. Another form of modification is the insertion of a marker gene in a region outside of but proximal to a gene of interest. This sort of modification results in the creation of a new linkage in the animal genome. For this approach, the precise function of a target sequence need not be known, so long as it is known to be associated with a particular trait. Selectable markers can be introduced into precise locations adjacent to desirable genes to facilitate selection of desirable traits that are otherwise not selectable in culture. This procedure is of value, for instance, in order to facilitate animal breeding programs. Segregation of the trait through successive generations can be tracked by growing cells on the appropriate selective medium. Thus, the time required to breed improved varieties can be shortened. As an example of this kind of approach, regions identified by RFLP analysis to be associated with complex traits can be targeted and cells containing the traits can be selected in culture.

(1) Targeting Construct

The targeting DNA comprises a rat sequence in which the desired sequence modifications are flanked by DNA sequence corresponding target sequence in the genome to be modified.

Preferably, the targeting DNA sequence is at least about 100-200 bp, more preferably at least about 300-1000 bp. The amount of targeting DNA present on either side of a sequence modification can be manipulated to favor either single or double crossover events, both of which can be obtained using the present invention. In a double crossover or "replacement-type" event, the portion of the targeting DNA between the two crossovers will replace the corresponding portion of the target DNA. In a single crossover or "insertion- type" event, the entire targeting DNA will generally be incorporated into the target sequence at the site of the single crossover. To promote double crossovers, the modification sequences are preferably flanked by targeting DNA such that, upon linearization, the modification sequences are located towards the middle of the flanking targeting DNA. If single crossovers are desired, the targeting DNA should be designed such that the ends of the linearized targeting sequence correspond to target DNA sequences lying adjacent to each other in the genome, as described by Thomas, K., and M. Capecchi, Cell 51:503-512 (1987).

The constructs can be made by PCR or cloned from different sources such as Pl library, bacteria lambda phage library, BAC library, or oligo synthesis. Targeting DNA contains at least a selectable marker. The selection methods based upon whole cell assays and which, preferably, employ a reporter gene that confers on its recombinant hosts a readily detectable phenotype that emerges only under conditions where a general DNA promoter positioned upstream of the reporter gene is functional. Generally, reporter genes encode a polypeptide (marker protein) not otherwise produced by the host cell which is detectable by analysis of the cell culture, e.g., by fluorometric, radioisotopic or spectrophotometric analysis of the cell culture.

Exemplary enzymes include esterases, phosphatases, proteases (tissue plasminogen activator or urokinase) and other enzymes capable of being detected by their activity, as will be known to those skilled in the art. More preferred for use in the present invention is green fluorescent protein (GFP) as a marker for transgene expression. The use of GFP does not need exogenously added substrates, only irradiation by near UV or blue light, and thus has significant potential for use in monitoring gene expression in living cells. As the previously existing selection procedures for identifying correctly modified cells required culture of the manipulated cells for 10-14 days in a chemical known as G418, it was necessary to pass the cells to fresh feeders during the selection procedure. However, the use of the green fluorescent protein (GFP) as an identification marker allows for identification of transgenic colonies without the need for passage or addition of selectable media. As a results the cells remain healthier and, since are not passaged repeatedly, maintain their ability to generate a living offspring after nuclear transfer or blastocyst injection.

Other preferred examples are the enzyme chloramphenicol acetyltransferase (CAT) which may be employed with a radiolabeled substrate, firefly and bacterial luciferase, and the bacterial enzymes .beta.-galactosidase and .beta.-glucuronidase. Other marker genes within this class are well known to those of skill in the art, and are suitable for use in the present invention.

(2) Transformation of Rat Stem Cells

Transformation of animal cells with the recombinant construct containing the targeting DNA can be carried out using essentially any method for introducing nucleotide sequences into animal cells including, as discussed below,

(a) Electroporation

In certain preferred embodiments of the present invention, the transgenic construct is introduced into the primordial germ cells via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge.

Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes, and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene in this manner.

It is contemplated that electroporation conditions for rat ES cells from different sources may be optimized. One may particularly with to optimize such parameters as the voltage, the capacitance, the time and the electroporation media composition. The execution of other routine adjustments will be known to those of skill in the art.

All of the rat ES cell lines from F344, Brown Norway, and hybrid have been successfully transformed using electroporation.

(b) Particle Bombardment

One of the preferred embodiments of the invention for transferring a naked DNA construct into cells involves particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. The microprojectiles used have consisted of biologically inert substances such as tungsten, platinum or gold beads.

(c) Viral Transformation

It is contemplated that the transgenic and targeting constructs can be delivered by the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. Adeno-associated virus (AAV) is an attractive vector system for use in the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells in tissue culture. AAV has a broad host range for infectivity, which means it is applicable for use with the present invention. Details concerning the generation and use of rAAV vectors are described in U.S. Pat. No. 5,139,941 and U.S. Pat. No. 4,797,368, each incorporated herein by reference.

The retroviruse infection can also be used. The retroviruses are a group of single- stranded RNA viruses characterized by an ability to convert their RNA to double- stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome. (d) Calcium Phosphate Co-Precipitation or DEAE-Dextran Treatment

In other preferred embodiments of the present invention, the transgenic construct is introduced to the cells using calcium phosphate co-precipitation. Mouse primordial germ cells have been transfected with the SV40 large T antigen, with excellent results. Human KB cells have been transfected with adenovirus 5 DNA using this technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-I, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990).

In another embodiment, the expression construct is delivered into the cell using DEAE- dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).

(e) Liposome Mediated Transformation

In a further embodiment of the invention, the transgenic construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self- rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is a transgenic construct complexed with Lipofectamine (Gibco BRL).

(3) Selection of Homologous Recombination Events

After the targeting DNA has been introduced into the animal cells, the cells in which the targeting DNA has stably integrated into the genome can be selected. The choice of which one to use will generally depend upon the nature of the sequence that has been integrated. For example, if the targeting DNA contains a selectable marker, as described above, then the integration of targeting DNA into the genome results in the stable acquisition of the selectable marker. In some situations the cells may be selected by virtue of a modification of the target gene. For example, if the target gene has a selectable phenotype, then modification of the target DNA may result in loss or alteration of that phenotype. In other situations, a selectable phenotype may result from juxtaposition of a DNA sequence present on the targeting DNA with DNA sequences present near the target DNA. For example, integration of a promoterless antibiotic resistance gene at the target site may result in expression of the resistance gene based on transcriptional activity at the target site.

It is also possible, although not essential, to use the polymerase chain reaction (PCR) to screen cells in which homologous integration has occurred. In an advantageous application, one PCR primer is directed to DNA in the modification sequence and another primer is directed to DNA near the target locus that is outside but proximal to the target DNA, such that integration results in the creation of a genomic DNA sequence in which the primer binding sites are facing each other in relative juxtaposition. After a number of rounds of amplification, DNA from such a locus will be present at much higher levels because it is being amplified exponentially rather than linearly.

Homologous recombination can be confirmed using standard DNA hybridization techniques, such as Southern blotting, to verify the presence of the integrated DNA in the desired genomic location.

Where the cells contain more than one copy of a gene, the cell lines obtained from the first round of targeting are likely to be heterozygous for the targeted allele. Homozygosity, in which both alleles are modified, can be achieved in a number of ways. One approach is to grow up a number of cells in which one copy has been modified and then to subject these cells to another round of targeting using a different selectable marker. Alternatively, homozygotes can be obtained by increasing the concentration of selecting reagent. Rat stem Cell DNA was re-hydrated with TE and collected into Eppendorf tubes. Phenol chloroform extraction was employed whenever necessary. Only samples with enough DNA for 3 Southern experiments were kept.

Five microgram of DNA was used for each sample. Each genomic cell DNA sample was digested by selected restriction enzyme at 37⁰C overnight. Units of restriction enzyme in each digestion was 200. After checking the completion of digest by electrophoresis, a 400 ml, 0.8% agarose gel was loaded with digested DNA samples and ran with molecular weight markers at 40V for 20 hr. The gel and running buffer was TBE containing ethidium bromide (0.5 μg/ml).

After electrophoresis is completed, photograph the gel. Place a transparent ruler alongside the gel so that the distance that any band of DNA has migrated can be read directly form the photographic image.

Capillary transfer was used to transfer DNA from agarose gels to nylon membranes. Trimmed gel was soaked for 30 minutes in 450 ml of alkaline buffer (1.5M NaCl, 0.5M NaOH) with constant and gentle agitation to denature the DNA. Rinse the gel briefly in deionized water, and then neutralize it by soaking for 30 minutes in 450ml of a solution of IM Tris (pH7.4), 1.5M NaCl at room temperature with constant, gentle agitation. Transfer buffer was 2OX SSC. Buffer was drawn from a reservoir and passes through the gel, membrane, into a stack of paper towels. The stack was left overnight.

After capillary transfer, the membrane was exposed to UV light to achieve cross-linking between DNA and the membrane. The membrane was then ready for hybridization.

"Church and Gilbert" buffer (7% (w/v) SDS, 0.5M Na-phosphate pH7.2, 1OmM EDTA, 0.25% BSA) was used for hybridization. Pre-hybridization of the membrane was carried out at 65⁰C in rotating oven for at least 1 hour. Hybridization probe was made with Rediprime II Random Prime Labelling System following manufacture's protocol. Hybridization was done in 65⁰C rotating oven overnight. Washes were done at 55⁰C. First was 4OmM sodium phosphate, 25 niM NaCl, 1% SDS, 1 mM EDTA, pH -7.2. Duration of the first wash was one hour. Second and third wash buffer was 0.1 ! SDS and 0.1 X SSC. The duration was 20 minutes each. Membrane was blot dry after wash and exposed to Kodak film. Duration of the exposure depends on radiation intensity detected by the beta-counter.

EXAMPLE 1

Preparation of Murine Embryonic Fibroblasts (MEF) for feeders

Three weeks prior to embryonic fibroblast isolation, a PGK-neo heterozygote male mouse is mated to a wild type C57BL/6 female. 13.5 days to 14.5 days after the plug is observed, sacrifice the pregnant female by CO2 asphyxiation.

Prepare three 10cm dishes containing 15ml PBS each. With the mouse on its back, wipe down the abdominal skin and fur with 70% alcohol. Make an incision down the midsection using scissors, thereby exposing the uterine horns. Observe how the embryos are located on each side of the uterus and in the amniotic sacs. Pull the embryos and uterine horns away from the abdomen and carefully detach them from the animal, placing them in a dish of PBS.

Using fine tipped scissors, cut open the uterine horns and release the embryos into the PBS. Next, carefully detach embryos from the amniotic sac and place in a fresh dish of PBS. While holding the embryo with a forcep, carefully pull out the liver, decapitate and rinse in another dish of fresh PBS, trying to get rid of as many red blood cells as possible.

Prepare 1 dish of Trypsin/EDTA, 15ml/dish. Place the decapitated embryos in the dish containing Trypsin/EDTA. Using curved Iris scissors finely mince the tissue quickly until it can be taken up in a 10ml pipette. Pipette up and down 6-7 times and place the dish of minced embryos in the incubator for 15 minutes. Add more Trypsin/EDTA to the dish and again pipette up and down with a 5ml pipette. Then place the dish back in the incubator for 10 more minutes.

Put all the contents of the dish in a 50ml conical tube. Allow the pieces of cellular debris to settle out over a period of 2 minutes. Remove the supernatant into a fresh tube, bring the volume up to 50ml with MEF media ( Dulbecco's Modification of Eagles Medium with 10% fetal calf serum, 1% penicillin / streptomycin, 1% L-glutamine) and spin at 1000 RPM for 5 minutes. Resuspend the pellet in MEF media and place the cells in a T- 150 flask. Let the cells grow overnight in the incubator. Leave the flask undisturbed for 24 hours. After this time, change the medium to remove any cellular debris and place your flask back in the incubator for another 24 hours.

This is the primary isolation or passage one. MEF's should attach and begin to divide in 1-3 days. During this time do not disturb, so as to allow MEF's to settle and attach.

After 2 days change the medium. It will be very acidic. After 3-4 days the culture will need splitting. Remove media and gently wash the monolayer with 2 X 10ml PBS. Add 2ml trypsin EDTA and split 1:4. After a further 2-4 days the culutre will be ready for freezing. The number obtained from each flask will be between 5-10 x 106 cells. Freeze cells in 10% DMSO at 3 X 106/ ampule.

When recovering the cells from LN2 put all the cells into a medium flask. When confluent these are split into 1 medium and one large flask (1:3). The large flask can be treated with mitomycinC and the medium flask split again. Do not passage beyond P6.

EXAMPLE 2

Generation of neonatal rat serum

Spague Dawley (Taconic) rats were used to produce neonatal rat serum. Blood was taken from the heart of new born rats, then put into eppendorf tubes and centrifuged at 4000 rpm for 8 min. The supernatant was taken out and put into a cryovial. All vials will be stored at -80 ⁰C. All manupilation should be done under the hood in case of the contamination.

EXAMPLE 3

Generation of neonatal rat liver conditional medium

Spague Dawley (Taconic) rats were used to produce neonatal rat liver conditional medium. Neonatal rat liver was taken from the new born rats and put into a sterile syringe with 17 gauge needle. Push the plunger and squeeze all liver tissue into 100 mm Petri dish with 4 ml culture medium containing 10v/v FBS (HyClone), 1 mM non-essential amino acids (GIBCO), 0.1 mM 2-mercaptoethanol (GIBCO), 1% pen-strep, 1% Glutamine. Then suck in all solution and tissue debis. Repeat twice and add 5 ml more culture medium and transfer all dishes into the incubator at 37 ⁰C with 5% CO_2. All medium in the dishes should be collected after three-day culturing and put into 50 ml Falcon tubes and kept at - 80 ⁰C freezer.

EXAMPLE 4

Establishment of rat embryonic stem cell lines and embryoid body generation

Establishment of rat embryonic stem cell lines: Embryos were flushed from the uterus of Brown Norway, Fisher344 and hybrid (Brown Norway X Fisher 344) females with FHM medium (Specialty Media) on the fifth day after natural mating (5 dpc). Well-developed blastocysts were transferred to ES medium in a 96-well plate with γ-irradiated mouse or rat embryonic fibroblasts as feeders. ES cell medium contains 15v/v FBS (HyClone), 1 mM non-essential amino acids (GIBCO), 0.1 mM 2-mercaptoethanol (GIBCO), 1% Glutamine, 1% pen/strep, 1000 units/mL Leukemia-inhibiting factor (LIF) (Chemicon: ESGl 107), 15 ng/ml human bone morphogenetic protein 4 (BMP4), 5% v/v neonatal rat liver conditional medium, 1% v/v neonatal rat serum. After 3-6 days, the central mass of each explant was trpsinized with 0.25% Trypsin/EDTA for 5 min. Resultant primary ES cell colonies were individually passaged into wells of a 96 well plate with feeders. Thereafter, cells were expanded by trypsinization of the entire culture (Figure 1 & 2).

Embryoid body generation: Rat ES cells were grown to near confluence, removed gently with 0.25% trypsin-EDTA solution and diluted to a concentration of IXlO⁵ cells/ml with ES cell medium. To separate small aggregates of ES cells from the embryonic fibroblasts, the suspension was left in the culture dish in the CO₂ incubator for 15 min for fibroblast attachment. Then, about ImI of the fibroblast-free suspension diluted with 9 ml of ES cell medium without LlF was added to 100-mm plates coated with 1% gelatin and incubated in the CO2 incubator. The medium was changed every 2-3 days. Embryoid bodies were formed in 7-9 days (Figure 3).

EXAMPLE 5

Construction of Targeting Vector and Gene Targeting in Rat Embryonic Stem Cells

Apolipoprotein All plays an important role in the high density lipoprotein (HDL) metabolism. HDL is also called good cholesterol. The apolipoprotein All gene has 4 exons. Exon 1 is nontranslated. The codoning starts from exon 2 and ends at exon 4. Targeting vector was constructed by using a 3.1 KB DNA fragment as short arm, which was PCR fragment from All 13 to AIIl 2. All 13 located 3.5 KB up stream of exon 2 containing start ATG, with a sequence of 5 '-TTCCTCATTCTCAAGACCTATGTCC-S '. All 12 located 380 BP up stream of exon 2, with a sequence of 5'- TCTTGCTGTCGCAGTGAACCACAG-3'. The short arm was inserted into 5' of Neo gene cassette using MIuI site. The long arm was a 4.2 KB PCR fragment from AII5 to AID. AII5 located 2.3 KB down stream of exon 4 containing stop codon, with a sequence of 5'-TCCCTGCTTCCAAGATAACCTTAGAC-3'. AID located 6.5 KB down stream of exon 4, with a sequence of 5 '-GTATCTTGACTTCTGTGGAAGTGG-S '. The long arm was inserted into 3' of the Neo cassette using a Bam HI site. In this strategy, the entire codoning region of apolipoprotein All was replaced by Neo gene cassette. Ten micrograms of targeting vector was linearized by Smal inside the short arm. It makes short arm becomes 2.2 KB to facilitate PCR screen. The vector was then transfected by electroporation of WH7 rat embryonic stem (ES) cells (Brown Norway). After selection in G418, surviving colonies were expanded, and Southern analysis was performed to identify clones that had undergone homologous recombination.

The Southern was done by probes from the 5' end outside of All construct and the 3' end outside of All construct. The probe from the 5' end outside of All construct was a 1.4 KB PCR product using primer pair A1IPB3 and AIIPB4. Primer AIIPB3 located 1.5 KB up stream of the Smal site, with a sequence of 5' -GGC AAGAACTCTGCCTC AGTTTCC- 3'. Primer AIIPB4 located 2 BP up stream of the Smal site, with a sequence of 5'- CTTCGATGAGCTGCACCGGTTGTG-3'. The probe from the 3' end outside of All construct was a 1.5 KB PCR product using primer pair AII3ENDPB1 and A1I3ENDPB2. Primer AII3ENDPB1 located 410 BP down stream of the AID, with a sequence of 5'- GACATTCTCCCAGGGTAGACAGAC-3'. Primer AII3ENDPB2 located 1.9 KB down stream of the AID, with a sequence of 5 '-GTAAACTGTTTGGGCCTGAGTGTC-S '.

For the 5' end outside probe (AIIPB3 and AIIPB4), Mouse genomic DNA was cut by Nsil. The wild type was 8.6 KB and knockout (KO) was 7.5 KB because of the newly introduced Nsil site in the 5' Neo gene cassette. For the 3' end outside probe (AII3ENDPB 1 and AII3ENDPB2), Mouse genomic DNA was cut by Spel. The wild type was 14 KB and knockout (KO) was 11 KB because of the newly introduced Spel site in the Neo gene cassette (Figure 4 & 5).

Claims

What is claimed is:

1. A method of growing rat embryonic stem cells, comprising growing a cell culture comprising rat embryonic stem cells from an embryo of a rat.

2. The method of claim 1, wherein said rats are from Brown Norway, Fisher 344 and hybrid (Brown Norway x Fisher 344) strains.

3. The method of claim 1, wherein said cell culture comprising rat embryonic stem cells is isolated from an embryo of said rats.

4. The method of claim 1, wherein said culture medium further comprises an effective amount of human bone morphogenetic protein 4.

5. The method of claim 1, wherein said culture medium further comprises an effective amount of leukemia inhibitory factor.

6. The method of claim 4, wherein said culture medium comprises leukemia inhibitory factor at a concentration of between about 5 ng/ml and about 100 μg/ml.

7. The method of claim 1, wherein said culture medium further comprises an effective amount of non-essential amino acids with respect to said rats.

8. The method of claim 6, wherein said culture medium comprises non-essential amino acids to said rat at a concentration of between about 10 nM and about 250 nM.

9. The method of claim 1, wherein said culture medium further comprises an effective amount of L-glutamine.

10. The method of claim 8, wherein said culture medium comprises L-glutamine at a concentration of between about 0.1 mM and about 50 mM.

11. The method of claim 1, wherein said culture medium further comprises an effective amount of beta-mercaptoethanol.

12. The method of claim 1, wherein said culture medium comprises neonatal rat serum.

13. The method of claim 1, wherein said culture medium comprises neonatal rat liver conditional medium.

14. A method of growing rat embryonic stem cells, comprising growing a cell culture comprising rat embryonic stem cells from an embryo of a rat on feeder cells for a time sufficient to obtain undifferentiated rat embryonic stem cells, said feeder cells at a density of between about 50 cells/cm. sup.2 and about 300 cells/cm.sup.2, in a culture medium comprising an effective amount of human bone morphogenetic protein 4 , said culture medium including exogenously added leukemia inhibitory factor.

15. A method of growing rat embryonic stem cells, comprising growing a cell culture comprising rat embryonic stem cells from an embryo of a rat on feeder cells for a time sufficient to obtain undifferentiated rat embryonic stem cells, said feeder cells at a density of between about 50 cells/cm.sup.2 and about 300 cells/cm.sup.2, in a culture medium comprising an effective amount of human bone morphogenetic protein 4 , said culture medium including neonatal rat liver conditional medium and neonatal rat serum.

16. A method for modifying the genome of the rat cells by homologous recombination between a target DNA sequence in the rat cell genome and a targeting DNA molecule introduced into the cell, said method comprising:

a) constructing a targeting DNA molecule in which a desired sequence modification is contained in a targeting segment of DNA

b) introducing said targeting DNA molecule into target cells; and

c) isolating a target cell in which said targeting DNA molecule is incorporated into its genome following homologous recombination between said targeting DNA molecule and said target DNA sequence thereby modifying the cell genome of said of said target cell.

wherein the introducing step and the isolating step are performed in vitro, and further provided that where the target cells are embryo-derived cells, embryo-derived cells are rat cells.

17. A method according to claim 16, wherein the rat cells are rat embryonic stem cells.

18. A method according to claim 16, wherein the desired sequence modification in the targeting DNA segment comprises an insertion of a selectable marker.

19. A method according to claim 18, wherein the selectable marker is a gene conferring resistance to a compound inhibitory to cell growth.

20. A method according to claim 18 wherein the selectable marker is a gene conferring the ability to grow on a selected substrate.

21. A method according to claim 18 wherein the selectable marker is a gene conferring the physical profile, such as GFP.

22. A method according to claim 16, wherein the desired sequence modification in the targeting DNA segment comprises an reporter gene.