Homologous Recombination In Embryonic Stem Cells Research Articles

Mouse Gene-Targeting Strategies for Maximum Ease and Versatility.

Well-planned strategies are an essential prerequisite for any mutational analysis involving gene targeting. Consideration of the advantages or disadvantages of different methods will aid in the production of a final product that is both technically feasible and versatile. Strategies for gene-targeting experiments in the mouse are discussed, including the rationale behind some of the common elements of gene-targeting vectors, such as homologous DNA and the use of different site-specific recombinases. We detail positive and negative selection as well as screening strategies for homologous recombination events in embryonic stem (ES) cells. For the planning stages of making different types of alleles, we first consider general strategies and then provide details specific to either homologous recombination in ES cells or making alleles by gene editing with CRISPR-Cas in preimplantation embryos. The types of alleles considered are null or knockout alleles, reporter gene knock-in alleles, point mutations, and conditional null alleles.

Targeted Mutations in the Mouse via Embryonic Stem Cells.

Genetic modification of mouse embryonic stem (ES) cells is a powerful technology that enabled the generation of a plethora of mutant mouse lines to study gene function and mammalian biology. Here we describe ES cell culture and transfection techniques used to manipulate the ES cell genome to obtain targeted ES cell clones. We include the standard gene targeting approach as well as the application of the CRISPR/Cas9 system that can improve the efficiency of homologous recombination in ES cells by introducing a double-strand DNA break at the target site.

Welcoming a new age for gene therapy in hematology

AbstractOur capacities to understand and manipulate mammalian genomes are accelerating at an astounding pace. In 2007, Capecchi, Evans, and Smithies were awarded the Nobel Prize in medicine for their work on gene targeting, which showed that embryonic stem cells could be modified by homologous recombination (HR) with engineered template DNA to alter virtually any gene and create mutant mice. This work revolutionized biology by allowing investigators to study the in vivo consequences of selected gene alteration. However, the efficiency of HR in embryonic stem cells is unpredictable, depending on the target gene and HR template. More importantly, spontaneous HR occurs at very low rates in most somatic cells, restricting the use of standard gene targeting for most laboratory and clinical applications. This limitation is being overcome by genome-editing technologies, which markedly enhance the capacity to alter cellular genes with laser-like precision. Four review articles in this edition of Blood summarize the field of genome editing, focusing on its potential for treating hematological disorders.

Homologous recombination efficiency enhanced by inhibition of MEK and GSK3β.

Homologous recombination in embryonic stem cells (ESCs) is widely utilized in genome engineering, particularly in the generation of gene targeted mice. However, genome engineering is often plagued by the problem of low homologous recombination efficiency. In this study, we developed a novel method to increase the efficiency of homologous recombination in ESCs by changing its culture conditions. By comparing the efficiency of different ESCs in various culture conditions, we determined that chemicals that inhibit the MEK and GSK3β pathways (2i condition) enhance homologous recombination and eliminate differences in efficiencies among cell lines. Analysis of gene expression patterns in ESCs maintained in different culture conditions has identified several homologous recombination-related candidates, including the pluripotent markers Eras and Tbx3. The results of this study suggest that homologous recombination is associated with ESC pluripotency.

Three Scientists to Share 2007 Nobel Prize in Physiology or Medicine

Three scientists—2 Americans and 1 from the United Kingdom—will share this year’s Nobel Prize in Physiology or Medicine. The new Laureates—Dr Mario R. Capecchi, Sir Martin J. Evans, and Dr Oliver Smithies—made a series of groundbreaking discoveries concerning embryonic stem (ES) cells and DNA recombination in mammals. Their discoveries in homologous recombination led to the creation of an immensely powerful technology referred to as gene targeting in mice, a technology that is now applied to virtually all areas of biomedicine, from basic research to the development of new therapies. Dr Capecchi is the Howard Hughes Medical Institute Investigator and Distinguished Professor of Human Genetics and Biology at the University of Utah, Salt Lake City, Utah. Born in 1937 in Italy, he received a PhD in Biophysics in 1967 from Harvard University. Sir Evans is the Director of the School of Biosciences and Professor of Mammalian Genetics, Cardiff University, United Kingdom. Born in 1941 in Great Britain, he received his PhD in Anatomy and Embryology in 1969 from University College in London. Dr Smithies, an American citizen, was born in Great Britain in 1925. He is the Excellence Professor of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill. He received a PhD in Biochemistry in 1951 from Oxford University. Drs Capecchi and Smithies both had the vision that homologous recombination could be used to specifically modify genes in mammalian cells. Capecchi demonstrated that homologous recombination could take place between introduced DNA and the chromosomes in mammalian cells, and showed that defective genes could actually be repaired by such homologous recombination. Smithies initially tried to repair mutated genes in human cells. He hypothesized that certain inherited blood diseases could be treated by correcting the disease-causing mutations in bone marrow stem cells. In these attempts, Smithies discovered that endogenous genes could be targeted irrespective of their activity, suggesting that all genes may be accessible to modification by homologous recombination. However, the cell types initially studied by Capecchi and Smithies could not be used to create gene-targeted animals. This would require another type of cell, one that could give rise to germ cells. Only then could the DNA modifications be inherited. Martin Evans had worked with mouse embryonal carcinoma (EC) cells, which, although originating in tumors, could give rise to almost any cell type. He had the vision to use EC cells as vehicles to introduce genetic material into the mouse germ line. His attempts were initially unsuccessful because EC cells carried abnormal chromosomes and could not therefore contribute to germ cell formation. Looking for alternatives, Evans discovered that chromosomally normal cell cultures could be established directly from early mouse embryos. These cells are now referred to as embryonic stem cells. The next step was to show that ES cells could contribute to the germ line. Embryos from 1 mouse strain were injected with ES cells isolated from another mouse strain. These mosaic embryos (composed of cells from both strains) were then carried to term by surrogate mothers. The mosaic offspring was subsequently mated, and the presence of ES cell–derived genes were detected in the pups. These genes would now be inherited according to Mendelian distribution. Evans then began to modify the ES cells genetically and for this purpose chose retroviruses, which integrate their genes into the chromosomes. He demonstrated transfer of such retroviral DNA from ES cells, through mosaic mice, into the mouse germ line. By 1986, all the pieces were at hand to begin generating the first gene-targeted ES cells. Capecchi and Smithies had demonstrated that genes could be targeted by homologous recombination in cultured cells, and Evans had contributed the necessary vehicle to the mouse germ line—the ES cells. The next step was to combine the two. For their initial experiments, both Smithies and Capecchi chose a gene (hprt) that was easily identified. This gene is involved in the rare inherited human disease Lesch–Nyhan syndrome. Capecchi refined the strategies for targeting genes and developed a new method that could be generally applied. The first reports in which homologous recombination in ES cells was used to generate gene-targeted mice were published in 1989. Since then, the number of reported knockout mouse strains has risen exponentially. Gene targeting has developed into a highly versatile technology. It is now possible to introduce mutations that can be activated at specific time points, or in specific cells or organs, both during development and in the adult animal. The Laureates received their award in Stockholm on December 10, 2007.

Evolutionarily Conserved Mammalian Adenine Nucleotide Translocase 4 Is Essential for Spermatogenesis

The adenine nucleotide translocases (Ant) facilitate the transport of ADP and ATP by an antiport mechanism across the inner mitochondrial membrane, thus playing an essential role in cellular energy metabolism. We recently identified a novel member of the Ant family in mouse, Ant4, of which gene configuration as well as amino acid homology is well conserved among mammals. The conservation of Ant4 in mammals, along with the absence of Ant4 in nonmammalian species, suggests a unique and indispensable role for this ADP/ATP carrier in mammalian development. Of interest, in contrast to its paralog Ant2, which is encoded by the X chromosome and ubiquitously expressed in somatic cells, Ant4 is encoded by an autosome and selectively expressed in testicular germ cells. Immunohistochemical examination as well as RNA expression analysis using separated spermatogenic cell types revealed that Ant4 expression was particularly high in spermatocytes. When we generated Ant4-deficient mice by targeted disruption, a significant reduction in testicular size was observed without any other distinguishable abnormalities in the mice. Histological examination as well as stage-specific gene expression analysis in adult and neonatal testes revealed a severe reduction of spermatocytes accompanied by increased apoptosis. Subsequently, the Ant4-deficient male mice were infertile. Taken together, these data elucidated the indispensable role of Ant4 in murine spermatogenesis. Considering the unique conservation and chromosomal location of the Ant family genes in mammals, the Ant4 gene may have arisen in mammalian ancestors and been conserved in mammals to serve as the sole and essential mitochondrial ADP/ATP carrier during spermatogenesis where the sex chromosome-linked Ant2 gene is inactivated.

Tissue-specific Changes in the Hydroxylysine Content and Cross-links of Collagens and Alterations in Fibril Morphology in Lysyl Hydroxylase 1 Knock-out Mice

We have generated mice with targeted inactivation of the Plod1 gene for lysyl hydroxylase 1 (LH1). Its human mutations cause Ehlers-Danlos syndrome VIA (EDS VIA) characterized by muscular hypotonia, joint laxity, and kyphoscoliosis. The Plod1(-/-) mice are flaccid and have gait abnormalities. About 15% of them died because of aortic rupture and smooth muscle cells in non-ruptured Plod1(-/-) aortas showed degenerative changes. Collagen fibrils in the Plod1(-/-) aorta and skin had an abnormal morphology. The LH activity level in the Plod1(-/-) skin and aorta samples was 35-45% of that in the wild type. The hydroxylysine content was decreased in all the Plod1(-/-) tissues, ranging from 22% of that in the wild type in the skin to 75 and 86% in the femur and lung. The hydroxylysylpyridinoline crosslinks likewise showed decreases in all the Plod1(-/-) tissues, ranging from 28 and 33% of that in the wild type in the aorta and cornea to 47 and 59% in femur and tendon, while lysylpyridinolines were increased. The hydroxylysines found in the Plod1(-/-) collagens and their cross-links were evidently synthesized by the other two LH isoenzymes. Few data are available on abnormalities in EDS VIA tissues other than the skin. Plod1(-/-) mice offer an in vivo model for systematic analysis of the tissue-specific consequences of the lack of LH1 activity and may also provide a tool for analyzing the roles of connective tissue in muscle function and the complex interactions occurring in the proper assembly of the extracellular matrix.

Correction of β Thalassemia by Homologous Recombination in Embryonic Stem Cells.

Genetic correction of patient-derived embryonic stem (ES) cells is a powerful strategy for the treatment of hemoglobinopathies such as β thalassemia and sickle cell disease. One genetic strategy for the correction of β thalassemia is to replace mutant or deleted β-globin alleles with a wild-type gene by homologous recombination in ES cells. Thalassemic mice that mimic the disorder have been generated by targeted gene deletion of the adult murine β-globin genes (PNAS 92: 9259–9263). We derived ES cells from our β-globin knockout mice and produced genetically identical mutant mice by injecting the ES cells into tetraploid embryos. These cloned β thalassemic mice have a severe microcytic anemia characterized by a marked reduction of the erythrocyte mean corpuscular volume (MCV), hemoglobin level (Hb), and hematocrit (Hct), and a marked increase in reticulocytes and red cell distribution width (RDW) compared to cloned wild-type control animals. In contrast to the normochromic, normocytic erythrocytes of wild-type clones, erythrocytes in peripheral blood smears of β thalassemic mice were hypochromic and exhibit extreme anisopoikilocytosis. A targeting construct containing 8.7 kb of mouse homology flanking a human γ- and β-globin gene cassette and a hygromycin marker gene was electroporated into the β thalassemic ES cells. After selection, DNA from 48 ES cell colonies was analyzed by PCR to identify homologous recombinants. Nineteen colonies (40%) had correctly integrated the human globin genes into the deleted mouse β-globin locus. Correctly targeted cells were injected into tetraploid blastocysts to produce mice that are derived solely from the corrected ES cells. These cloned mice synthesize high levels of human β-globin polypeptide that corrects the α- to β-globin chain imbalance, thereby eliminating the thalassemic erythrocyte morphology. The MCV, Hb, Hct, RDW, and reticulocyte levels in the blood of these mice are normal. These results demonstrate that a severe hemoglobinopathy can be cured after targeted gene replacement of a mutant gene(s) with a wild-type allele by homologous recombination in ES cells.

Engineering embryonic stem cell derived glia for adenosine delivery

Based on the anticonvulsant and neuroprotective properties of adenosine, and based on the long-term survival potential of stem cell derived brain implants, adenosine releasing stem cells may constitute a novel tool for the treatment of epilepsy. Pluripotency and unlimited self-renewal make embryonic stem (ES) cells a particularly versatile donor source for cell transplantation. With the aim to test the feasibility of a stem cell-based delivery system for adenosine, both alleles of adenosine kinase (ADK), the major adenosine-metabolizing enzyme, were disrupted by homologous recombination in ES cells. Adk −/− ES cells were subjected to a glial differentiation protocol and, as a result, gave rise to proliferating glial precursors, which could be further differentiated into mature astrocytes and oligodendrocytes. Thus, a lack of ADK does not compromise the glial differentiation potential of ES cells. The Adk −/− ES cells yielded glial populations with an adenosine release of up to 40.1 ± 6.0 ng per 10 5 cells per hour, an amount considered to be sufficient for seizure suppression. Our findings indicate that Adk −/− ES cells constitute a potential source for therapeutic adenosine releasing grafts.

Chemical mutagenesis of the mouse genome: an overview

The careful comparison of the phenotypic variations generated by different alleles at a given locus, including of course, those alleles with a deleterious effect, is often an important source of information for the understanding of gene functions. In fact, every time it is possible to match a specific alteration observed at the genomic level with a particular pathology, it is possible to establish a relationship between a gene and its function. When considered from this point of view, the production of new mutations by experimental mutagenesis appears as an alternative to the strategy of in vitro gene invalidation by homologous recombination in embryonic stem (ES) cells, with the advantage that experimental mutagenesis does not require any previous knowledge of the gene structure at the molecular level. Homologous recombination in ES cells is a 'gene driven' approach, in which mutant alleles are produced for those genes that we already know. Experimental mutagenesis, on the contrary, is a 'phenotype driven' approach, in which unknown genes are identified based on phenotypic changes. Also, while homologous recombination in ES cells requires a rather sophisticated technology, mutagenesis is simple to achieve but relies greatly on the efficiency of the mutagenic treatment as well as on the use of an accurate protocol for phenotyping. In this review, we will address a few comments about the different techniques that can be used for the induction of point mutations in the mouse germ line with special emphasis on chemical mutagenesis. We will also discuss the limitations of experimental mutagenesis and the necessity to look for alternative ways for the discovery of new genes and gene functions in the mouse.

Targeted Disruption of a Murine Glucuronyl C5-epimerase Gene Results in Heparan Sulfate Lacking l-Iduronic Acid and in Neonatal Lethality

The glycosaminoglycan, heparan sulfate (HS), binds proteins to modulate signaling events in embryogenesis. All identified protein-binding HS epitopes contain l-iduronic acid (IdoA). We report that targeted disruption of the murine d-glucuronyl C5-epimerase gene results in a structurally altered HS lacking IdoA. The corresponding phenotype is lethal, with renal agenesis, lung defects, and skeletal malformations. Unexpectedly, major organ systems, including the brain, liver, gastrointestinal tract, skin, and heart, appeared normal. We find that IdoA units are essential for normal kidney, lung, and skeletal development, albeit with different requirement for 2-O-sulfation. By contrast, major early developmental events known to critically depend on heparan sulfate apparently proceed normally even in the absence of IdoA.

Proteoglycan gene targeting in somatic cells.

Gene targeting allows the generation of specifically designed mutations in cells by the mean of homologous recombination between exogenous DNA and endogenous genomic sequences (-). This is possible because a fragment of genomic DNA introduced into a cell can locate and recombine with the chromosomal homologous sequences (). Although the mechanisms of exogenous DNA transfer to the nucleus and of homologous recombination are still poorly understood, important advances have been made in identifying the requirements for the targeting vector and the desirable features of the targeted locus that maximize gene targeting (). Presently, various gene targeting techniques can be successfully applied to a variety of organisms. In mammals, gene targeting strategies have been developed mainly for the genetic manipulation of mouse embryonic stem (ES) cells. Mating of the transgenes leads to the generation of animals in which the functional significance of a specific gene silencing can be investigated in different conditions. Cell lines can also be established from mouse organ biopsies. There are, however, important limitations to the attainability of cell lines of interest from transgenic animals. First, ES gene knockout techniques are currently limited to the mouse. Because there are major interspecies differences with regard to gene pattern of expression and implication in disease, extrapolation from the results obtained with mice cells to the pathophysiological mechanisms in humans is not warranted. This is particularly the case when genes involved in malignant transformation and development are investigated. Second, the availability of established cell lines bearing specific mutations makes somatic gene targeting highly desirable. In this case the implication of the expression of a particular gene on the cell behavior can be assessed directly against a well-defined genetic background. Third, the establishment of an organ-specific cell line involves complex genetic rearrangements leading to immortalization. In other words, cell lines, even if derived from the same animal cannot be considered isogenic in the sense that the pattern of expression of several genes - as opposed to the single targeted gene-is affected. In addition, the genetic mutation that has been introduced through homologous recombination in ES cells may be lethal even at a stage preceding embryonic development, thus precluding the possibility of establishing a cell line. Finally, the cost of originating and maintaining a transgenic animal colony is high.

Chimeric Mouse Production by Microinjection

The culmination of the creation of a mutation in mouse embryonic stem (ES) cells is, commonly, the generation of a mouse line that can propagate the mutation. The ability to combine methods of homologous recombination in ES cells with blastocyst-mediated transgenesis has resulted in an explosion of tailored mutant mouse strains. These animals provide research tools that are virtually impossible to create using other methodologies. This unit describes the methods necessary to generate chimeras, from injection of the ES cells into the blastocoel cavity of 3.5-day-old embryos through the implantation of the injected embryos into the foster mother. The resultant pups are true chimeras: their tissues are derived from both the host embryo and from the ES cells. If the ES cells are able to populate the germ line, the chimera can pass an altered gene to offspring, resulting in a new mouse strain in which all cells contain an altered gene.

Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.

Hormone-sensitive lipase (HSL) is known to mediate the hydrolysis not only of triacylglycerol stored in adipose tissue but also of cholesterol esters in the adrenals, ovaries, testes, and macrophages. To elucidate its precise role in the development of obesity and steroidogenesis, we generated HSL knockout mice by homologous recombination in embryonic stem cells. Mice homozygous for the mutant HSL allele (HSL-/-) were superficially normal except that the males were sterile because of oligospermia. HSL-/- mice did not have hypogonadism or adrenal insufficiency. Instead, the testes completely lacked neutral cholesterol ester hydrolase (NCEH) activities and contained increased amounts of cholesterol ester. Many epithelial cells in the seminiferous tubules were vacuolated. NCEH activities were completely absent from both brown adipose tissue (BAT) and white adipose tissue (WAT) in HSL-/- mice. Consistently, adipocytes were significantly enlarged in the BAT (5-fold) and, to a lesser extent in the WAT (2-fold), supporting the concept that the hydrolysis of triacylglycerol was, at least in part, impaired in HSL-/- mice. The BAT mass was increased by 1.65-fold, but the WAT mass remained unchanged. Discrepancy of the size differences between cell and tissue suggests the heterogeneity of adipocytes. Despite these morphological changes, HSL-/- mice were neither obese nor cold sensitive. Furthermore, WAT from HSL-/- mice retained 40% of triacylglycerol lipase activities compared with the wild-type WAT. In conclusion, HSL is required for spermatogenesis but is not the only enzyme that mediates the hydrolysis of triacylglycerol stored in adipocytes.

Homologous gene targeting to study ECM assembly.

Genetically engineered mutant strains of mice have become invaluable too to decipher complex biological processes, such as ECM assembly, and to dissect phenotypic overlaps, like the genodermatoses. Homologous gene targe ing in embryonic stem (ES) cells is increasingly favored over the transgen approach because it allows the study of mutations in homozygosity and without altering the remainder of the genome (1). Homologous gene targeting has been successfully employed to abrogate the expression or alter the structure of ECM-coding genes (2,3). In both cases, the experimental protocol consists of the following three steps: a. construction of the targeting vector; b. homologous recombination in ES cells; and c. generation of mutant mouse strains.

Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice.

Fibroblast growth factor-2 (FGF-2) has been implicated in various signaling processes which control embryonic growth and differentiation, adult physiology and pathology. To analyze the in vivo functions of this signaling molecule, the FGF-2 gene was inactivated by homologous recombination in mouse embryonic stem cells. FGF-2-deficient mice are viable, but display cerebral cortex defects at birth. Bromodeoxyuridine pulse labeling of embryos showed that proliferation of neuronal progenitors is normal, whereas a fraction of them fail to colonize their target layers in the cerebral cortex. A corresponding reduction in parvalbumin-positive neurons is observed in adult cortical layers. Neuronal defects are not limited to the cerebral cortex, as ectopic parvalbumin-positive neurons are present in the hippocampal commissure and neuronal deficiencies are observed in the cervical spinal cord. Physiological studies showed that FGF-2-deficient adult mice are hypotensive. They respond normally to angiotensin II-induced hypertension, whereas neural regulation of blood pressure by the baroreceptor reflex is impaired. The present genetic study establishes that FGF-2 participates in controlling fates, migration and differentiation of neuronal cells, whereas it is not essential for their proliferation. The observed autonomic dysfunction in FGF-2-deficient adult mice uncovers more general roles in neural development and function.

Escherichia coli gpt as a positive and negative selectable marker in embryonal stem cells

Transfection of HPRT − L fibroblasts with a plasmid containing two linked selectable markers genes, gpt and neo, regulated by the same eukaryotic control elements, yielded a 6-fold higher transfection frequency on selection for neo than for gpt. Transfection of HPRT − embryonal stem (ES) cells with the same plasmid yielded high levels of transfectants when selected for neo expression with G418, but a level of transfection greater than two orders of magnitude lower was observed when HAT supplemented medium was used to select for gpt expression. Selection for gpt expression in ES cells with medium containing mycophenolic acid and xanthine gave slightly higher frequencies of transfection, but still considerably lower than that for neo selection. In addition, mycophenolic acid exhibited a general cytotoxicity to ES cells with the window between toxicity of this compound to gpt − ES cells and gpt + ES cells being very narrow. Cells selected with mycophenolic acid and xanthine for expression of gpt remained sensitive to HAT selection. Expression of gpt in a representative ES cell line, selected on mycophenolic acid and xanthine, was verified by Northern analysis and sensitivity to 6-thioguanine. While the level of mRNA expression in this ES cell line was insufficient to support growth via purine salvage when exposed to HAT medium, identical levels of gpt expression in HPRT − L cells, as judged by Northern analysis, allowed for normal growth in HAT medium. This suggests that ES cells place a greater demand on purine nucleotide biosynthesis than L cells. These results are discussed in terms of the use of gpt as a positive and negative selectable marker for gene targeting via homologous recombination in ES cells.

Transgenic animals: techniques.

The methodology for generating transgenic mice has opened up new vistas in genetic manipulation of the mammalian genome. Transgenic mice can be generated by injection of DNA into the zygote pronucleus (Palmiter and Brinster 1986) retroviral infection of early embryos (Soriano et al. 1986) or introduction of DNA into pluripotent embryonic stem (ES) cells in culture followed by colonization of the germ line in chimeras (Robertson et al. 1986). Most of the work utilizing transgenic mice has involved defining cis-acting regulatory elements that control tissue-specific and developmental expression of genes. This is still an important area of research and two of the workshop presentations dealt with such studies: one involving use of an in situ detectable reporter gene construct for analyzing tissue-specific gene expression and the other, the role of long-range sequences in controlling gene expression. However, transgenic animals are increasingly being considered as tools for manipulating endogenous gene expression to investigate gene function. Two possible approaches, use of antisense RNA or targeted mutagenesis, were described. Transgenic mice also offer the possibility of obtaining new mutations by insertional mutagenesis and may also be used to identify new genes active in embryogenesis by integration of reporter constructs. Dr. Lesley Toth described studies on the regulation of the expression of one of the mouse homeo-box genes, Hox-3. I. When 5' regions of this gene were linked to the Escherichia coli P-galactosidase gene and used to generate transgenic mice, tissue localization of gene expression could be detected by staining with X-gal (Zakany et al. 1988). Blue staining cells were detected in a region of the developing spinal cord similar to that defined for the endogenous gene by in situ hybridization. Expression outside this region was detected, suggesting that not all control sequences were included in the construct. Further definition of the spinal cord controlling region is underway. Two common observations in transgenic mice are that level of transgene expression is not directly related to copy number and varies with site of integration. Dr. Lee Wall described how, for the P-globin locus, such effects can be overcome by inclusion of sequences located at DNAse I superhypersensitive sites removed from the close vicinity of the gene (Grosveld et al. 1987). These sequences may allow formation of chromosomal domains that protect the transgene from effects due to integration sites. Whether such sequences flank other genes is not yet known. The possibility of manipulating gene expression by injection of antisense RNA into eggs was discussed by Dr. Bob Erickson. His group has shown that antisense P-glucuronidase RNA injected in large excess into mouse zygotes can significantly suppress P-glucuronidase enzyme activity throughout preimplantation development (Bevilacqua et a1 . 1988). Similar studies utilizing antisense nRNA to one of the gap junction protein genes are underway, and effects on uncoupling of cell communication have been observed. This approach has potential for investigating the role of several genes active in the early embryo. A more definitive means of analyzing the function of any known gene in development is to mutate the gene and study the phenotype of the mutation. Dr. Tom Doetschman described the development of targeted mutagenesis in mammals by homologous recombination in pluripotent embryonic stem cells. Once a mutation has been produced in the gene of interest, the stem cells can be injected into blastocysts to generate chimeric mice which will hopefully contain germ cells derived from the embryonic stem cells and thus carry the mutant gene. By this means, a mutation induced in culture can be transferred into the mouse stock for genetic analysis. Dr. Doetschman has demonstrated the feasibility of such approaches by both curing HPRT(Doetschman et al. 1987) mutations and generating HPRTmutations by homologous recombination in ES cells. Strategies for applying this approach to other genes were discussed and are underway in a number of laboratories. Dr. Janet Rossant described a novel approach to identifying new genes active in embryogenesis which is very similar to an approach being developed in Drosophila. Vectors containing the E. coli lacZ gene driven by a weak promoter were introduced to ES cells, and a number of clonal lines were used to generate chimeras. Analysis of P-galactosidase expression in midgestation revealed that the lacZ gene could be activated in specific spatial patterns, indicating that cis-acting regulatory elements close to the site of integration were activating the gene. By choosing the most interesting patterns of expression and cloning out the host gene that activates lacZ, it should be possible to identify a new set of genes important in embryonic development. Discussion at the workshop demonstrated that this whole area of genetic manipulation of the mammalian gene line has a lot of potential for elucidating problems in all aspects of mammalian genetics.

Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells

The deletion mutation in the HPRT-deficient mouse embryonic stem (ES) cell line E14TG2a has been corrected by gene targeting. The presence of plasmid sequences in the correcting vector DNA did not affect the frequency of correction. We have characterized three different HPRT gene structures in correctants. Cells from one corrected clone have been introduced into mouse blastocysts, and germ line transmission of the ES cell-derived corrected gene has been achieved. The corrected gene has the same pattern of expression as the wild-type gene, with the characteristic elevated level of expression in brain tissue. Hence, we have demonstrated the feasibility of introducing targeted modifications into the mouse germ line by homologous recombination in ES cells.