Reverse genetics using transgenic mice.

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REVERSE GENETICS USING TRANSGENIC MICE Carlisle P. Landel, Shizhong Chen, and Glen A. Evans Molecular Genetics Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037

KEY WORDS;

toxigenes, cell ablation, Thy-I, homologous recombination, embryo injection

INTRODUCTION Genetics provides a powerful tool for the analysis of development. Pro karyotes and lower eukaryotes that are amenable to genetic analysis have provided useful model systems for the dissection of complex physiologic processes. Traditional genetic analysis utilizing these organisms involves the creation of mutations that are selected on the basis of phenotype and used to understand the nature of the underlying genotype. This type of mutational genetics as applied to mammals and higher eukaryotes has been severely limited because of the difficulty in obtaining sufficient mutants for complete developmental analysis. In recent years, however, the use of transgenic organisms, in which pseudo-mutations are implanted in the germline through micromanipulation, has suggested a genetic approach in which the traditional logic is reversed. In this reverse genetic approach, a genotype is designed and constructed in vitro and implanted in the mouse germline by microinjection or by transfection into embryonic stem cells. The resulting transgenic animals often display a phenotype dependent on the particular design of the mutated gene. This reverse genetic approach allows a designed genotype to be used to discover the resulting pheno type and is particularly applicable to the analysis

of mammalian development. Using transgenic technology, mutations can be produced to result in (a) aberrant expression of otherwise normal genes, (b) targeted ablation of cell populations, and (c) insertional inactivation of genes by homologous recombination. 841

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LANDEL, CHEN & EVANS

MUTATIONS THAT RESULT IN ABERRANT GENE EXPRESSION Mutations that are likely to provide important information of developmental introduction of genes containing various

significance may result from the

tissue-specific regulatory elements that can redirect the tissue-specific expres sion of normal gene products. This type of reverse genetic genotype places the


expression of a normal gene under the control of regulatory elements that will

regulation of gene expressi on. One example which is now widely used, is the expression of oncogenes under the control of tissue-specific promoters to induce malignant transformation in the target tissue (21). alter the temporal or spatial

.

A second approach is to express a gene of unknown function or de velopmental significance according to an altered developmental program. In this method the resulting phenotype may suggest how the gene product functions in normal development. The Thy-l glycoprotein is a 25-kd cell surface protein that is an important lineage marker in mouse T lymphocytes. The protein sequence suggests that the Thy-l antigen gene, immunoglobulin genes, and other immunoglobulin-superfamily genes evolved from a common primordial gene, and its conservation of expression in vertebrates and some invertebrates suggests a functional importance. In spite of much speCUlation,

is unknown. Thy-l is expressed on mature T lymphocytes, thymocytes, hematopoietic stem cells, and most central neurons, and its expression can be induced on activated B lympho

however, the function of the Thy-l antigen

cytes by treatment with interleukin-4. Since antibodies reacting and cross linking the Thy- l antigen induce T cell receptor-mediated activation of T cells, Thy-l is thought to play a role in T cell or progenitor cell activation and maturation. Reverse genetics was used to determine if Thy-l might be important for lymphocyte activation or maturation through an attempt to alter the normal pattern of Thy- l expression in transgenic mice

(5, 16). A hybrid Thy-l gene

was constructed in which the immunoglobulin heavy chain enhancer was

inserted into a large intron located downstream of the Thy-l promoter region. Transgenic mice were produced by microinjecting the normal and altered Thy-l

gene into isolated mouse embryos and several founder animals were

obtained. Transgenic animals carrying the normal Thy-l gene expressed the gene at physiologic levels on thymocytes and in the brain, with slightly lower levels on splenic T cells. Animals carrying the Thy-1!EJ.t construction also expressed Thy-Ion the surface of mature B lymphocytes and pre-B cells as

by the coexpression of the B cell surface antigen B220. In addition to bone marrow cells ex pre ssed Thy-I, as compared to 7% or less Thy-l + cells in normal bone

judged

the expression of Thy-Ion B lymphocytes, over 80% of

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marrow. These transgenic mice developed a pre-B cell nonmalignant hyper plasia of the bone marrow and lymph nodes where the predominant cell types express surface characteristics of both T and B lymphocytes and are similar in many ways to the naturally occurring lprllpr mutant mouse. Further analysis suggests that the presence of Thy-Ion the surface of pre-B cells induces rapid proliferation in vivo and in long-term bone marrow


cultures. These data tend to suggest that Thy-l may be involved in a signal

transducing mechanism for the regulation of growth and expansion of lym phocyte progenitors in response to a lymphokine or other growth factor, or cell-cell interaction. Redirecting the expression of a gene to an inappropriate tissue in transgenic mice, then, can yieJd a phenotype that is extremely informative when dissecting the function of a new gene product. This approach is dependent upon understanding the regulation of tissue-specific gene expression and the availability of

appropriate controlling elements.

MUTATIONS THAT RESULT IN PROGRAMMED CELL DEATH Programmed cell death is an important aspect of mammalian development and occurs as a critical aspect of development of the mammalian nervous and immune systems. A few rare naturally occurring mutations have been dis covered that result in premature or inappropriately timed cell death and subsequent developmental abnormalities. The past few years have seen the development of techniques for targeted cell ablation in transgenic animals by the expression of toxic gene products in a tissue-specific or developmentally stage-specific manner (for reviews, see 1, 8). This strategy involves the construction of "toxigenes" where a tissue-specific regulatory sequence is used to drive the expression

of a gene encoding a toxic polypeptide. Initially

silent, when activated at the appropriate developmental stage, expression of the toxin in the target cell induces cell suicide. This technique has proven useful for the investigation of developmental lineages, in determining the significance of cell-cell interactions in development, and in examining the functions

of individual cell types

.

ablation studies: diptheria and ricin. Diptheria toxin is a single 62-kd polypeptide produced by the tax gene carried by lysogenic corynephage of patho genic strains of Corynebacterium diptheriae. which is responsible for human diptheria. It consists of two separable domains, a 22-kd A subunit and a 40-kb B subunit, which together account for the high level of toxicity for most mammalian cells. The B subunit binds to the cell surface and mediates internalization of the A subunit. When inserted into the cytoplasm, the A subunit catalyzes the ADP-ribosylation of elongation factor 2, which results in an immediate Two toxic polypeptides have been used for cell

toxin

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cessation of protein synthesis and rapid cell death. Ricin is a toxic lectin


produced by the castor bean Ricinus communis and exists as a heterodimer with noncovalently linked A and B subunits analogous to those of diptheria toxin. The B subunit contains a cell surface binding domain that recognizes surface galactose and induces internalization of the A subunit where it cata lyzes the cleavage of the adenosine at position 4365 from the phosphodiester backbone of the 28S rRNA. Inactivation of the ribosomal 28S RNA leads to the cessation of protein synthesis and rapid cell death. The construction of toxigenes requires in vitro genetic engineering of natural toxin genes. First, the coding sequence must be altered such that only a single toxic A subunit is made (18). Ricin is produced as a single nontoxic propeptide from which the A and B subunits are cleaved by proteolysis. Th e leader polypeptide must be removed to prevent secretion and allow cytoplasmic expression of the toxigene. Finally, suitable 3' and 5' flanking sequences, including restriction sites for insertion of appropriate regulatory sequences, must be included. The initial demonstration of this technique used the 205 base pair 5' flanking region of the pancreatic elastase gene fused to the diptheria toxin A subunit toxigene (DT-A). Elastase is an important digestive protease pro duced exclusively by the acinar cells of the pancreas and represents one of the m ajor protein products of differentiated pancreatic acinar cells. Transgenic mice were produced carrying the pancreas-specific toxigene by direct microinjection into isolated embryos (20). Expression of this toxigene at an early stage of embryogenesis would be expected to eliminate, at the very least, cells that are committed to differentiation into exocrine pancreatic cells by virtue of the e arly expression of elastase. As a result of these initial studies, seven of 24 founder transgenic animals carrying the elastase-DT-A toxin developed normally with the exception of severe abnormalities of the pan

was associated with a severe reduction in the number of islet and ductal cells and a virtual absence of exocrine cells. Ablation of the pancrease at an early stage of development led to early death of the animals, making survival and derivation of a strain of tran sgeni c mice for further study difficult. Toxigen es have been used effectively for the an alysis of development of the crystallin lens in transgenic mice through the development of DT-A and ricin A (R-A) vectors. Crystallins are produced by developing lens fiber cells and are encoded by a large multigene family. The genes encoding the major forms of crystallins, er, {3, and y, are differentially regulated during lens mor phogen esi s and produced by terminally differentiated lens fiber cells in large quantities. In the case of both the erA-crystallin gene and the y2 crystallin gene,S' flanking sequences and promoters appear to contain the majority of regulatory signals for precise temporal and spatial coordination of gene expression. Moreover, alterations in differentiation pathways and elimination

creas. The expression of this gene construct

-

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of lens precursor cells do not affect viability or breeding potential of resulting transgenic mice so that strains may be established through breeding. Breitman et

al (4) constructed transgenic mice in which the y2-crystallin promoter al

directed the expression of DT-A in transgenic mice. Similarly, Landel et

(15) expressed the R-A toxigene from an aA-crystallin promoter and derived strains with well-defined abnormalities of lens development. Both aA crystal


lin-R-A and 12 crystallin-DT-A mice demonstrate a profound microphthalmia characterized by fluid filled vesicles replacing much of the normal adult lens. Transgenic mice expressing the y2-crystallinlDT-A toxigene demonstrated lens abnormalities whose severity varied among both different strains and individuals within a single strain ranging from cataracts and mild structural abnormalities to near destruction of the lens. The aA-crystalliniricin animals demonstrated lens abnormalities and, in addition, a malformation and abnor mal development of the neural retina. In addition, these animals produced ectopic lens fiber cells suggestive of

a

trans determination of neural retina to

lens. Toxigene expression using pituitary-specific regulatory signals has been useful for clarifying some aspects of cell determination in the developing anterior pituitary. The precise lineage relationship between growth hormone producing somatotropes and prolactin-producing lactotropes is unknown. A 310 bp

5' flanking sequence of the rat growth hormone gene is sufficient to

direct expression to somatotropes and their immediate progenitors and has been used to direct cell ablation using a toxigene construction (2). Fusion of the growth hormone promoter with the DT-A toxigene resulted in three of 21 founder mice that lacked detectable levels of circulating growth hormone and demonstrated impaired growth. These animals had a nearly complete absence of somatotropes in the pituitary. In the transgenic pituitary, the 200,000 somatotropes were reduced in number to an average of 10 cells. Since earlier evidence had suggested that a r are population of pituitary producing cells both growth hormone and prolactin represented a common progenitor of somatot ropes and lactotropes examination of pituitaries from these transgenic an ,

imals could be used to test this hypothesis. Accompanying the absence of somatotropes was a severe decrease in the number of lactotropes, which suggested a developmental relationship between the two. The authors also noted rare islands of growth hormone producing cells, however, which suggested that some cells apparently escaped the expression or effects of the toxigene in the ablated pituitary. An alternate approach to the expression of substances that are toxic is the targeted expression of genes that are not of themselves toxic, but which can

metabolize drugs to toxic substances. The use of sensitizing genes rather than toxic genes 'may allow the production of viable animals where targeted ablation may be carried out in a tissue or cell type that is critical for normal development or survival. The herpes virus thymidine kinase

(HSV-tk) is not

846


harmful to most terminally differentiated cells and is widely used as a that have been d ev elo ped for viral chemotherapy can be converted by the viral thymi

selective marker in tissue culture studies. Nucleoside a nalogu es

dine kinase to toxic metabolites. Acyclovir, PIAU [1-(2-deoxy-2-fluoro-/3-D arabinofuranosyl)-5-iodouracil], gancyclovir, and related drugs arc relatively non-toxic to mammalian cells at low doses but block the replication of herpes

(9). These drugs are not metabolized by mammalian thymidine kinases, but are phosphorylated by HSV-tk to nucleoside monophosphates, which can be further metabolized to nucleoside triphosphates by host enzymes, which


virus

leads to an inhibition of DNA synthesis. Transgenic mice have been generated in which the HSV-tk gene is expressed in a cell type specific manner, and in which cell ablation occurs after treating the animals with these drugs (3, 3a, 10). A

fusion of the HSV-tk gene with the immunoglobulin kappa light chain

promoter and immunoglobulin heavy chain enhancer was used to generate transgenic mice. These regulatory elements direct gene expression in lym

phoid c ells of spl een, thymus, bone marrow, and lymph nodes in transgenic mice. Founder animals were derived that express detectable HSV -tk activity

in spleen and thymu s and low or undetectable activity in most other tissues Upon treatment of these animals with gancyclovir, a dramatic reduction in the .

number of hematopoietic cells was seen that was directly related to the drug level in the blood. Severe atrophy of the spleen and lymph nodes was seen, which reduced the number of lymphoid cells to 15% of normal. Cell pop ulations in the thymus were reduced to 2% of the normal number of thymo cytes, with a virtual absence of the cortex and an extremely hypocellular medulla. Removal of the drug allowed almost complete repopulation of most lymphoid lineages, which indicated that nondividing progenitor cells were essentially not affected (10). Thymidine kinase obliteration was also directed to the anterior

pituitary (3a). HSV-tk was placed under the control of the or prolactin (Prl) promoter to ablate somatotropes and

growth hormone (GH)

lactotropes, respectively. Treatment of the GH-HSV-tk mice with FIAU resulted in dwarf mice that essentially lacked both somatotropes and lactot ropes, again suggesting that these cells share a common progenitor. Removal of the drug allowed repopulation of both cell types, which indicates that stem cells persist in the adult animal. Treatment of the PrI-HSV-tk mice with PIAU had no effect, thus indicating that PrJ expression and lactotrope differentia tion are post-mitotic events. Unlike DT-A or R-A toxigenes that inhibit

protein synthesis, the thymidine kinase obliteration technique inhibits DNA synthesis, and thus ablation depends almost en ti rely on cell division in the target tissue. This approach is most applicable to tissues where cells are dividing rapidly through the adult life of the animal, as in the immune system.

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MUTATIONS THAT MODEL HUMAN DISEASES Transgenic animals that display phenotypes similar to human disorders may be useful for dissecting pathogenesis, for defining gene function, and for testing modalities of therapeutic intervention. The generation of animal mod els for human disease has taken several approaches including the insertion of genes encoding mouse homologs of human dominant mutations into the


mouse germJine in attempts to create a mouse analogue of a human disorder; the insertion of a human gene carrying a dominant mutation as a transgene to express a visible phenotype; and the insertion of the gene from a pathogenic virus into the mouse germline to simulate the pathogenesis associated with infection. Osteogenesis imperfecta

(01) type 2 is a autosomal dominant disorder

caused by the substitution of a single glycine residue in the triple helix of the al-procollagen gene

COLlAl. This substitution in a repeating Gly-X- Y

structure alters the molecule so that collagen assembly is affected and results in abnormalities of bone formation. A similar phenotype was induced in a transgenic mouse strain by performing in vitro mutagenesis on the mouse homolog of the human 0'1 procollagen gene to induce the same amino acid

substitution. The mutated gene was then used to produce transgenic mice by microinjection into isolated mouse embryos (23).

All of the resulting

transgenic animals died shortly after birth due to severe developmental defects in the formation of the skeleton. Analysis of these mice for the mutant procollagen demonstrated that expression of the mutation gene at levels as low as 10% of normal was sufficient to disrupt bone formation and lead to death. This suggests that the defective protein inhibits collagen assembly rather than forming a less stable molecule that is susceptible to degradation or altered secretion. In addition, this study demonstrates that human disorders may be modeled using transgenic animals by engineering suitable mutations in vitro. Dycaico et al

(7) used a human gene carrying a dominant mutant allele to

create an animal model for the neonatal hepatitis of human a)-antitrypsin

deficiency. arantitrypsin is a serum protein that inhibits trypsin, elastase, thrombin, and other serine proteases and prevents destruction of alveolar walls which, in the absence of a normal functioning enzyme, leads to emphysema. Associated with this disorder induced by the Z allele is hepatitis caused by failure of the abnormal a)-antitrypsin to be correctly transported across

the

endoplasmic

reticulum.

About

15%

of

neonates

that

are

homozygous for this allele develop hepatitis and obstructive jaundice and cirrhosis, presumably associated with defects in protein transport. The mutant human Z allele was used for the construction of transgenic mice and the resultant founders expressed human

a I-antitrypsin

at approximately the same

levels as found in humans. The transgenic animals also accumulated the


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mutant protein in the liver and, presumably because of faulty transport, developed hepatitis similar to that seen in human neonates. Thus these animals provide an appropriate model system for a dominant human disorder by expressing the human allele. Various viral pathogens exist whose range is limited to humans and whose pathogenesis is poorly understood. One use of reverse genetics using transgenic mice is to create models of pathogenesis induced by these viruses by directly inserting all or part of the viral genome into transgenic mice, thus bypassing many restrictions in host range of the virus, which prevent the use of animal models. IC virus (lCV) is a human papovavirus that induces a multifocal leukoencephalopathy in humans characterized by chronic de myelination of nerves. Expression of the JCV early region after insertion into the genome of transgenic mice induces a similar disease state involving widespread demyelination and neurological symptoms similar to the natural mouse mutations jimpy and quaking (22, 25). The phenotype of these mice suggests that the expression of ICV T antigen arrests the maturation of oligodendrocytes and inhibits myelin production. Human T-Iymphotropic virus type 1 (HTLV-l) is suspected to be a causative agent of adult T-cell leukemia and has been associated with neurologic disorders such as spastic paraperesis and multiple sclerosis. The tat gene of this virus encodes a transactivator of viral and host gene expression. Mice transgenic for the HTLV-1 tat gene express the gene product at high levels in nervous tissue. These animals develop tumors reminiscent of von Recklinghausen's neurofi bromatosis in muscle and in the thymus, which results in thymic depletion and growth retardation (12, 19). The host range of human immunodeficiency virus HIV is limited to humans and chimpanzees, but it causes acquired immunodeficiency syndrome only in humans. Models for the disease are being sought via the production of mice transgenic for part or all of the HIV genome. Transgenic mice carrying the HIV tat gene develop a syndrome similar to Kaposi's sarcoma, which is seen as the initial manifestation of AIDS in about 25% of patients and which eventually develops in about 50% of all patients. Since the HIV tat gene is a transactivator of transcription, like the HTLV-I tat gene, these data suggest that the HIV tat gene may directly induce the malignancies seen in AIDS patients through the activation of endogenous proto-oncogenes. HIV itself can not infect mice, as mice do not produce the HIV receptor CD4. In fact, HIV will not infect murine cells transfected with the CD4 gene and producing the protein on their surface appears to block viral internalization. To circumvent this block and infect mice directly, mice were made transgenic for the HIV genome (17, 26). These mice produce an infectious virus, develop a syndrome that mimics many of the symptoms of AIDS, and die at 25 days of age.

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MUTATIONS THAT ELIMINATE THE EXPRESSION OF GENES Most reverse genetic mutations involve the gain of function or aberrant gene expression and simulate natural dominant mutations. The process of chromo somal insertion after injection of DNA into the embryo, however, will involve


random insertional inactivation of endogenous genes. About 15% of these insertions may result in a visible phenotype (usually embryonic lethal) when bred to homozygosity.

At a low but detectable frequency, however,

homologous recombination of exogenous DNA into the mammalian genome takes place. Homologous recombination resulting in an insertional inactiva tion mutation provides a technology for producing recessive mutations in transgenic mice. Gene inactivation involves the construction of an artificial gene such that insertion by homologous recombination will inactivate the endogenous gene. Transformation of embryonic stem (BS) cell cultures with this construction followed by screening for the desired insertional event can result in inactiva tion of the endogenous genc carried by one chromosome. After amplification of the transfected ES cell line, ES cells are injected into isolated blastocysts and following reimplantation will result in chimeric animals where the ES cells contribute to most somatic and germ cells. If germ cell chimerism is obtained, the animals may be bred to obtain the desired insertional mutation in a homozygous state. This approach of targeted mutagenesis has been used to inactivate the gene encoding the hypoxanthine-guanine phosphoribosyl transferase (HPRT) car ried on the X chromosome of mice. Insertional inactivation was accomplished by infection with a retrovirus and selection of ES cell clones that grow in HAT selective medium (14). When used to produce chimeric animals through blastocyst injection, animals with an inactive HPRT gene were produced. Targeted mutagenesis by homologous recombination has also been used to produce HPRT- chimeras (6, 11, 24). For most genes, a positive selection for gene inactivation is not available or easily derived. In these cases, a selection system can be included in the targeting vector, or ES cells in which homologous recombination has taken place can be rapidly screened using the polymerase chain reaction (PCR). The construction used for insertion is prepared to contain unique sequences, such as the neomycin/kanamycin phosphotransferase of tn9 (neof), and to poten tially interrupt expression of a gene after homologous insertion. The presence of neof allows cells containing a transfected gene to be selected by growth in the neomycin analogue geneticin (G418). For rapid screening, PCR primers are chosen so that only those ES cells in which homologous integration has

850

LANDEL, CHEN & EYANS

occurred will demonstrate an appropriately sized amplified fragment. Since

peR reactions can be automated, screening thousands of colonies to identify the appropriate insertion is now possible, Targeted mutagenesis has been used to create insertional inactivation muta

(l3, 27). Since these genes are Drosophila that are important for the control of develop

tions of the Hoxl.l, Hox1.2, and En-2 genes related to genes of


ment, the availability of mice with targeted mutations at these loci will provide important insights into the control of mammalian development.

CONCLUSIONS Traditional genetics depends on the creation of new mutations or the identification of naturally occurring mutations based on their phenotype. The detailed study of phenotype then allows the discovery and characterization of the mutant genotype and an eventual understanding of the underlying biology. In non-traditional or reverse genetics, the logic of experimentation proceeds backwards. One may now conceive of a possible genotype, prepare the genotype in the laboratory, and implant it into the mammalian genome to reveal the resulting phenotype. The advent of reverse genetics allows the creation of a wide variety of mutations that could not easily be obtained using traditional genetics and, with continued technical development and refine ment, promises to greatly expand our understanding of mammalian biology.

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REVERSE GENETICS 11. H ooper, M., Hardy, K., Handy sid e, A., H unter, S., Monk, M. 1987. HPRT d efic ien t (Lesc h-Nyh an) mouse embryos derived from germline colonization by cultured cells. Nature 326:292-95 12. Hinrichs, S. H . , Nerenberg, M., Reynolds. R. K. , K h oury, G., Jay, G. 1987 . A transgenic mouse model for hu man neurofibromatosis. Science 237; 1340-43 13. Joyuer, A. L., Skarnes, W. C, Rossan t , J. 1989. Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells. Nature 338:153-56 14. Kuehn, M. R., Bradley, A., Roberts on, E. J., Eva ns, M. J. 1987. A p ote nt ial animal model for Lesch-Nyan syndrome through introduction of HPRT mutations into mic e. Nature 326:295-98 15. Landel, C. P., Zhao, 1., Bo k, D., Evans, G. A. 1988. Lens-specific ex pression of recombinant ricin induces developmental defects in th e eyes of transgenic mice. Genes Devel. 2:116878 16. LandeJ, C. P., Chen, S., B ot teri, F., va n der Putten, H . , Evans, G. A. 1989. H ematop oietic abnormalities induced by ectopic expr essi on of the Thy-I antigen in trangenic mice. In Gene Transfer and Gene Therapy, ed. L Verma, R. Mul ligan, A. Beaudet, pp. 179-88. New York: Liss 17. Leona rd, J. M., Abramczuk, J. W., Pezen, D. S., Rutledge, R . , Belcher, J. H., et at. 1988. Development of disease and virus recovery in transg enic mice containing HIV proviral DNA. Science 242:1665-70 18. Maxwell, L H. , Maxwell, F., G lode, L. M. 1986. Regulated expression of a diphtheria toxin A-chain gene trans fected into human cancer cells: a possi ble strategy for inducing cancer cell su ic id e. Cancer Res. 46:4660-64

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