904 THE NEW ENGLAND JOURNAL OF MEDICINE April 4, 1996 MOLECULAR MEDICINE haploid genome can be passed on to subsequent gen- erations. KNOCKOUT MICE The goal of the gene-targeting (knockout) method is to replace the specific gene of interest with one that is JOSEPH A. MAJZOUB, M.D., inactive, altered, or irrelevant. To increase the probabil- AND LOUIS J. MUGLIA, M.D., PH.D. ity that such replacement will occur, rather than non- specific random integration of the DNA, both ends of the HE ability to remove or alter with precision a sin- replacement gene are flanked by long DNA sequences Tgle one of the thousands of genes in the body and homologous to the sequences flanking the target gene to transmit this mutation to all subsequent progeny was (Fig. 1). Gene constructs of this type permit corre- a science-fiction dream only a few years ago. But today sponding stretches of DNA to be exchanged (in what is this technique is part of a routine procedure for creat- termed “homologous recombination”) when the DNA ing animal models that can be used to study the patho- breaks and rejoins. The frequency of homologous re- physiology and therapy of diseases in humans. combination is low. Therefore, there must be a way of In general, mutations that cause a gain of function selecting the rare cells in which the target gene has been produce disease even when they occur in only one of a replaced by the constructed gene. Two selectable mark- gene’s two alleles; for example, the oncogenic muta- ers meet this purpose. tions that cause abnormal cell proliferation. In a reces- Figure 1 shows the use of selectable markers in an sive genetic disorder, by contrast, there must be muta- experiment using a genetically engineered knockout tions in both alleles for the disease to be produced, and vector to inactivate the gene that encodes corticotropin- the mutations cause a loss of function; for example, in releasing hormone (CRH) in mice. The vector was cre- patients with cystic fibrosis two copies of a recessive al- ated by replacing the gene encoding the CRH polypep- lele cause a loss of chloride-channel activity. tide with a gene that confers resistance to the antibiotic The methods needed to produce animal models of neomycin. This bacterial gene lies between the 59 and recessive genetic diseases differ from those used in study- the 39 homologous flanking regions present in both the ing autosomal dominant diseases. Integrating an onco- target and the replacement gene. The knockout vec- gene that causes dominant disease, such as c-myc, into tor also contains a gene that encodes viral thymidine the genome of a fertilized mouse oocyte without alter- kinase, which confers sensitivity to ganciclovir. This ing the mouse’s own genes creates a transgenic, cancer- second selectable marker lies outside the 59 and 39 ho- prone mouse that transmits this trait to its offspring mologous flanking regions of the replacement gene. with a dominant pattern of inheritance. To create an an- The genetically engineered DNA is introduced into imal model of an autosomal recessive disease, however, embryonic stem cells that are then incubated with tis- both alleles of the normal gene must be inactivated. sue-culture medium containing neomycin and ganciclo- The technique of gene “knockout” was developed for this purpose. Several independent scientific advances have cul- Figure 1. Homologous Recombination between a Cellular Gene minated in the ability to alter a single heritable gene in and a Knockout Vector to Create Mice Lacking Corticotropin- the DNA of a mouse with precision. Initially, chimer- Releasing Hormone (CRH), a Major Hypothalamic Regulator of ic mice with somatic cells from more than one genetic the Stress Response. background were created, though inefficiently, by intro- Embryonic stem cells (upper left-hand panel) contain the CRH cellular gene (upper right-hand panel), which consists of exon ducing embryonal carcinoma cells into normal mouse 1 (olive green, a 59 noncoding region), an intron, and exon 2 (red, embryos early in gestation. In the early 1980s, the ef- a protein-coding region, and yellow, a 39 noncoding region). A ficiency of the process used to generate chimeras im- knockout vector, consisting of a collinear assembly of a DNA proved markedly when methods were developed to flanking segment 59 to the cellular gene (blue), the phosphoglyc- culture totipotential cells from the inner cell mass of erate kinase–bacterial neomycin gene (pgk-neo, violet), a 39 seg- ment of the cellular gene (yellow), a DNA flanking segment 39 to the blastocyst, the area destined to become the fetus. the cellular gene (green), and the phosphoglycerate kinase–viral These pluripotential cells, termed embryonic stem cells, thymidine kinase gene (pgk-tk, orange), is created and introduced can be genetically altered and then microinjected into into the embryonic-stem-cell culture. Double recombination oc- the cavity of an intact mouse blastocyst after 3.5 (of a curs between the cellular gene and the knockout vector in the 59 homologous regions and the 39 homologous regions (dashed total of 19.5) days of gestation. They can, from the lines), resulting in the incorporation of the inactive knockout vec- blastocyst stage, populate all the tissues of the develop- tor, including pgk-neo but not pgk-tk, into the cellular genomic lo- ing mouse. The contribution of the embryonic stem cus of the embryonic stem cell. The presence of pgk-neo and the cells to the genetic makeup of the chimeric animal that absence of pgk-tk in these replaced genes will allow survival of develops from the injected blastocyst is most easily as- these embryonic stem cells after positive–negative selection with neomycin and ganciclovir (see text). sessed by using embryonic stem cells and blastocysts In the bottom panel, the clone of mutant embryonic stem cells is whose genes for coat color differ. If the embryonic stem injected into a host blastocyst, which is implanted into a pseu- cells contribute to the germ cells (for example, the dopregnant foster mother, and subsequently develops into a chi- sperm) of the developing mouse embryo, their entire meric offspring. The contribution of the embryonic stem cells to the germ cells of the chimeric mouse results in germ-line trans- mission of the embryonic-stem-cell genome to offspring that are From the Division of Endocrinology, Children’s Hospital, Harvard Medical heterozygous for the mutated CRH allele. The heterozygotes are School, Boston. mated to produce mutant mice homozygous for CRH deficiency, 1996, Massachusetts Medical Society. with impaired hormonal responses to multiple stressors. This document was created with FrameMaker 4 0 4 Vol. 334 No. 14 MOLECULAR MEDICINE 905 vir. Embryonic stem cells that have incorporated the outside the region of specific homologous recombina- new DNA by homologous recombination resist neo- tion (negative selection). mycin (positive selection). By contrast, cells that have In embryonic stem cells that survived this “positive– nonspecifically taken up genetically engineered DNA, negative” selection, the CRH knockout vector had cor- including the viral thymidine kinase, are killed by ganci- rectly replaced one of the normal CRH alleles. These clovir because of the gene for this kinase, which lies clones were then injected into blastocysts to initiate the Embryonic stem cell CRH gene 59 homologous Intron 39 homologous region region Cellular gene Embryonic-stem- cell culture Plasmid Homologous pgk-neo pgk-tk DNA recombination Knockout vector Cellular gene replaced Selection by neomycin and ganciclovir Injection of Implantation of chimeric embryonic stem cells blastocyst in foster mother into host blastocyst Germ-line offspring Chimeric offspring 906 THE NEW ENGLAND JOURNAL OF MEDICINE April 4, 1996 creation of chimeric mice (Fig. 1). Chimeras with germ Table1. Human Disorders Studied in Knockout Mice. cells derived from the altered embryonic stem cells trans- mitted the changed embryonic-stem-cell genome to their DISORDER TARGET MOUSE GENE OR GENE PRODUCT offspring, yielding mice heterozygous for CRH deficien- Cardiology cy; the heterozygotes were then bred to each other to Atherosclerosis Apolipoprotein E, low-density–lipo- protein receptor create mice homozygous for CRH deficiency. Salt-sensitive hypertension Atrial natriuretic peptide The deficits present in a knockout mouse can reveal Endocrinology or clarify the function of the mutant gene. The analysis Familial hypocalciuric hypercalcemia Calcium receptor by targeted inactivation of the genes implicated in fetal Glycogen storage disease type 1 Glucose-6-phosphatase development and organogenesis has been especially pow- Intrauterine growth retardation Insulin-like growth factor II Obesity b -Adrenergic receptor erful. Gene knockout has shown that the gene SF-1 3 (encoding steroidogenic factor 1) is essential for adrenal Gastroenterology Hirschsprung’s disease Endothelin receptor and gonadal organogenesis. Also essential are WT-1 Ulcerative colitis Interleukin-2 (the Wilms’ tumor locus), for renal development, and the Hematology genes encoding myogenin (for skeletal-muscle forma- a-Thalassemia a-Globin tion), the glucocorticoid receptor (for glucocorticoid- Hemophilia A Factor VIII mediated fetal development), and the estrogen receptor Chronic granulomatous disease Cytochrome b-245 (for female sexual maturation). RAG-2 (recombination- Immunology Autoimmune lymphoproliferative syn- FAS activating gene 2) and GATA-1 (which recognizes a drome GATA-nucleotide motif) are critical to the development