Transgenic animals S. Steven Potter Children's Hospital Research Foundation, Cincinnati, Ohio 45229, USA Current Opinion in Biotechno[ogy 1990 1:159-165
Introduction Because of the pioneering efforts of a number of investigators, it is now possible to manipulate the mammalian genome by a powerful array of methods. The ~¢orkhorse procedure of the past decade has been the pronuclear microinjection technique, first published by Gordon et al. [1]. This approach allows one to add genes to the genome efficiently, producing a mouse, for example, that carries new genetic information in every cell of its body. Pronuclear microinjection has been, and will continue to be, useful for a wide variety of experiments. Another technique uses homologous recombination in embryonal stem (ES) cells to produce mice with endogenous genes modified in a very precise manner. This ability to target changes in specific genes promises to be an important tool in the functional analysis of the mammalian genome. The biological impact of genetic alterations can be observed directly in the phenotypes of the transgenic animals, thereby yielding insight into a number of biological processes. Transgenic studies of carcinogenesis and the immune system have been reviewed recently [2 o]. Here, I will focus primarily on the advances made in the last year or so in using transgenic animals to study mammalian development.
Pronuclear microinjection Mammalian genome manipulation techniques generally involve the initial modification of a single cell which is then used to produce a mouse. For the pronuclear microinjection method, this cell is the zygote, or fertilized egg. The technique is conceptually straightforward, although the underlying molecular mechanisms of transgene insertion remain poorly understood. A few hundred copies of the gene construct to be added to the genome are simply microinjected into the male (larger) pronucleus a few hours before its fusion with the female pronucleus. Remarkably, in a significant fraction of these onecell embryos, some of the added DNA will covalently integrate into a single, random genomic position by a process of illegitimate (non-homologous) recombination. The in-
serted foreign DNA is referred to as a transgene, and it generally consists of a head-to-tail concatemer of a variable number of injected molecules. The injected cells are then simply implanted into the oviducts of surrogate mothers and 3 weeks later mouse pups are born. Generally at 2-3 weeks of age, their DNA is analyzed to determine which of them are actually transgenic (usually about 10-30%). Transgenic mice were originally used almost exclusively for gene expression studies [3]. In many respects, they represent an ideal in vivo expression system for the analysis of ctX-acting controlling elements associated with genes. DNA constructs lacMng putative control elements, or carrying elements connected to a reporter gene, can be tested for activity in these mice, where all cell types and all developmental times are available for analysis. Despite the disadvantages of cost, technical difficulty, and the variability of expression pattems resulting from different integration sites, this approach nevertheless remains powerful and is still often used. This is because, for many genes, an appropriately expressing cell type is not readily grown in culture, and in any event it is clear that cell culture gene assay systems do not recognize all of the control elements necessary for appropriate in vivo expression [4]. These transgenic mice are remarkably unperturbed by the presence within their cells of a promoter that is often driving high-level production of some bacterial reporter gene-encoded protein. The completely normal appearance of these mice suggests a considerable vigor within the system which can readily tolerate significant loads of innocuous foreign protein. With care, however, it is possible to design transgenes that are biologically active and alter the phenotype of the mouse in an informative way.
Dominant homeobox transgenes The homeobox genes were originally found in Drosop hila, where it was observed that a number of developmentally important genes carry a highly conserved 180bp sequence encoding a DNA-binding domain [5 °]. These genes have an important role in controlling
Abbreviations ES~embryonal stem; ho---hotfoot; HPRT--hypoxanthine phosphoribosyltransferase;IGF~insulin-like growth factor; Igl--legless; Id~limb deformity; PCR--polymerasechain reation. (~) Current Biology Ltd ISSN 0958-1669
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Mammaliangene studies Drosophila development by regulating the expression of other h o m e o b o x genes as well as 'subservient' batteries of genes. Some of the homeobox genes help to establish the pattern of segmentation; others determine segment identity. Some of these genes are apparently master switches that control the developmental destinies of groups of cells. An Antennapedia mutation, for example, can cause legs to form where the antennae would normaUy be. The homeobox is so evolutionarily well conserved that it has been possible to retrieve over 35 genes with extensive homology from the murine genome, some of which have now been studied in detail. Expression patterns of murine homeobox genes during development have been determined primarily by in situ hybridization to non-transgenic embryos, although the use of [~-galactosidase reporter transgenes is becoming increasingly popular, for example in localizing DNA control elements that drive region-specific expression [6.]. The results of these investigations are extremely interesting, and certainly suggest that the murine homeobox genes have a role in development; nevertheless, the evidence remains circumstantial. Without any known mutations in these genes, their true biological functions remain uncertain. Wolgemuth et al. [7] were the first to functionally analyze the murine h o m e o b o x genes (Iqox genes) by generating transgenic mice; they used a DNA construct that included the H o x l . 4 gene with 10kb of upstream DNA and an SV40 sequence tag at the 3' end to distinguish transcripts from those of the endogenous gene. In situ hybridization revealed that the transgenic mice, which had extra copies of Hoxl.4, expressed higher levels of transcripts, particularly in the gut. Interestingly, this resulted in a developmental abnormality, termed megacolon, in which the bowel is grossly distended and feces are not easily extruded from the distal end of the colon. These experiments were the first to demonstrate a developmental malformation resulting from inappropriate expression of a mammalian h o m e o b o x gene. Bailing et al. [8".] carried this approach one step further when they made transgenic mice with the chicken [3-actin promoter connected to the coding sequence of Hoxl. 1. This heterologous promoter produced almost ubiquitous low-level expression of the Hoxl.1. gene, allowing a variety of tissues to be tested for a possible developmental response. The resulting mice were indeed malformed, with craniofacial abnormalities including open eyes at birth, detached pinnae and cleft secondary palate. The mice invariably died within 14 days of birth. Careful analysis of the more severely affected mice also revealed some intriguing abnormalities in the development of the cervical somites [9"'].
Dominant transgenes and human disease A number of extremely interesting dominant transgenes that promise to increase our understanding of certain human diseases deserve a mention. For example, it is
now possible to generate mice that produce human hemoglobin [10-.,11 ..], and with further refinements it appears likely that an excellent animal model for sickle cell disease will result. In another study, Heckel el al. [12 ..] have produced mice that overexpress urokinasetype plasminogen activator, and consequently suffer a severe bleeding disorder. Finally, Scott et al. [13"] have demonstrated the importance of prion protein in determining scrapie susceptibility, incubation time and neuropathology by producing transgenic mice that express the hamster prion gene.
Insertional mutations A useful by-product of the promoter assay and other types of transgenic mouse experiments described above is the generation of insertional mutations in endogenous genes. That is, in a small fraction (about 5%) of transgenic lines, the transgene will insert by chance into a chromosomal position that results in the inactivation of the gene originally residing there. Such mutations are particularly useful because of the molecular handle provided by the known transgene sequences, which allows molecular retrieval and analysis of the disrupted gene. Furthermore, the genes identified by insertional mutagenesis are known to be interesting from the outset, as one begins with a mutant mouse displaying a phenotype resulting from the inactivation of the gene. A number of interesting insertional mutations have recently been better described than before or reported for the first time. The limb deformffy (ld) mutation, first characterized by Woychik et al. [14], is certainly among the most developmentally fascinating. They found that mice homozygous for the transgene insertion displayed a phenotype that included synostoses (fusion) of the long bones of both forelimbs and hindlimbs, oligodactyly and syndactyly of the hand and foot bones, and a high frequency of unilateral and bilateral renal aplasias. This mutation was shown to be aUelic to two previously found spontaneous ld alleles. In a careful embryologic analysis of ld/ldmutants, Zeller et al. [15 " ] have found more recently that limb buds appear defective as early as day 10, with a shortened anterior-posterior axis and an apical ectodermal ridge that never achieves the correct morphology. At later times in development, it was shown that the ulna and radius form as separate elements that then fuse abnormally, whereas the tibia and fibula actually form as a single unit. Moreover, molecular analysis of this locus is now revealing an intriguing story of a very large gene that gives rise to several distinct transcripts during development. The insertional mutation named legless (lgl) also affects limb development [16]. The mutant mice (lgFlgi) are born with no hindlimb structure distal to the femur. Furthermore, they lack anterior distal portions of the forelimbs and anterior portions of the brain. In a more detailed description of the newborn mutant phenotype, McNeish et al. [17.] mention another interesting char-
Transgenic animals Potter acteristic. They observed that 50% have situs inversus, (with inverted viscera). Chromosome mapping results and complementation tests strongly suggest that this insertional mutation is allelic with a classic murine mutation locus. Mouse DNAs flanking the transgene insertion have been recovered and their molecular analysis is underway (Singh, Supp, McNeish, Copeland, Jenkins and Potter, unpublished data). Krulewski etal. [18..] have described an insertional mutation in which homozygotes have a number of fascinating neurologic defects, including a total absence of Purkinje cells, degeneration of photoreceptor ceils from the retina, and a loss of almost all mitral cells from the olfactory bulb. Most of the homozygote males (18 out of 22) were also sterile, with histologic analysis revealing no sperm in the epididymis. This insertional mutation was shown to be allelic with the previously described classic mutant locus, pcd ( Purkinje cell degeneration), which exhibits a similar phenotype. Molecular analysis of this locus is in the initial stages, with mouse DNA apparently flanking the transgene insertion retrieved, but with no coding sequences yet identified. Remarkably, Gordon's group has also identified another insertional mutation that affects both neurologic development and spermatogenesis [19"]. The homozygotes show an ataxic gait when about 2 weeks old, the severity of which increases with age. Histologic analysis revealed no detectable neurologic abnormalities. The homozygous mice were functionally sterile and, although sperm counts were normal, the mutant males were shown to produce sperm defective in their ability to penetrate the egg zona. Interestingly, this insertional mutation, too, was found to be allelic with a previously identified 'classic' mutation in the mouse genome termed ho~oot (ho). Once again, flanking sequences abutting the transgene insert have been retrieved and molecular analysis has begun. Another insertional mutation resulting in male sterility has been described by MacGregor et al. [20.]. In this case, the homozygous mice are apparently normal in all other respects, although developing spermatids do not mature properly. This mutation, named sys, is not allelic with any previously characterized locus. Xiang etal. [21 ..] have described an insertional mutation resulting in mice that are much reduced in size. Matings showed that this 'mini-mouse' insertional mutation is allelic with the spontaneous mutation pygmy. These mice are proportionately correct, except for short ears, and have normal circulating levels of growth hormone and somatostatin. It is to be hoped that molecular analysis of the disrupted gene will lead to a better understanding of comparable human dwarf syndromes. It is clear that the careful screening of transgenic lines for the presence of possible recessive insertional mutations provides a powerful method for randomly scanning the murine genome for genes of particular interest. Mthough this approach is certainly yielding dividends, there are often serious ditficulties. First, in some cases the transgene is highly methylated and as a result is very refractory to cloning in almost all Escherichia coli strains, which
are capable of recognizing foreign methylation patterns and selectively destroying DNA. Cells that lack both the McrA and McrB systems still restrict some transgenes, but as new cells are now available that are more completely restriction-deficient (DIt10B), this problem is disappearing. A second and more common difficulty results from the complexity of the transgene insertion. It is often, for example, found associated with a deletion of genomic DNA at the insertion site, which can range in size from very small (less than 1 kb), to very large (more than 100 kb). Extensive deletions obviously mean that a large region must be scrutinized for the presence of the gene of interest. Perhaps even more perplexing, however, are the cases where the transgene is actually mixed with murine genomic DNA 'islands'. These islands, which can also vary in size, greatly complicate the analysis. It is clear that the degree of chromosomal destruction rendered by the transgene insertion must be carefully assessed, so that all possible candidate genes can be evaluated. On the positive side, however, the molecular tag of the transgene insertion will almost always allow the investigator to retrieve a mouse genomic sequence that is at least 'close' (estimated as within 100kb) to the dismpted gene. Although this will still leave much work to be done, it nevertheless represents an excellent starting point, at least by the standards of those who do 'reverse genetics' or cloning based on chromosomal position. Also, it is becoming clear that a surprisingly large fraction of insertional mutations are allelic with previously described 'classic' mutations. This can be extremely useful in the identification of the gene of interest, as one can compare wild-type, classic mutant and insertional mutant transcripts. The final diagnostic tests, however, are still rescue of the wild-type phenotype by adding back a wildtype copy of the gene into transgenic mice, or knock-out of the gene in wild-type mice, using ES cells, to generate the phenotype independently.
Enhancer traps, gene traps, and retroviral insertions The enhancer trap approach [22,23..] has both advantages and disadvantages when compared with the standard insertional mutagenesis described above. This technique uses a ]3-galactosidase gene connected to a weak promoter as a transgene. On occasion, the expression patterns of such a transgene which are readily detected by Xgal (5-bromo-4-chloro-3-indoyl-B-D-galactopyranoside) staining, are particularly interesting. So with this approach one begins the molecular analysis knowing something of the expression pattern, but lacking a phenotype. It must be noted that interesting expression patterns are not always associated with interesting gene function. Moreover, the molecular search for the gene is further complicated by the ability of enhancers to work from considerable distances, thus expanding the genornic region that must be analyzed.
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Mammalian gene studies The gene trap method in some ways combines the best of the enhancer trap and standard insertional mutagenesis techniques. In this method, a promoterless, but spliceacceptor-pre-fixed [3-galactosidase gene is used, and insertion into an endogenous gene can result in expression. Hence, one begins the molecular analysis with knowledge of the expression pattern, an excellent molecular handle, and probably a functionally disrupted gene. Insertional mutations can also be generated by retroviral infection of mouse embryos or ES cells [24,25]. In this method, one copy of the retroviral genome integrates 'cleanly', with no deletion or scrambling of host genomic DNA. This approach has been used effectively in the past and still holds great promise for the future. The very real advantage it offers is the relative ease of molecular characterization of the insertional mutations.
Gene targeting Although the pronuclear microinjection procedure is quite effective for adding DNA concatemers to random genomic locations, there are many problems that are better approached by targeted modification of endogenous genes. For example, in the cases of the many cloned genes for which there are no known classic mutations, inactivation and/or modification of endogenous copies of these genes would greatly facilitate a functional analysis. This can now be achieved by homologous recombination in ES cells. ES cells are derived from the inner cell mass of a blastocyst, and can be cultured for prolonged periods and yet retain totipotency [26]. That is, when reintroduced into a blastocyst they can contribute to all cell lineages, including the germ line, of the resulting embryo. The general scheme for targeted gene modification of ES cells is illustrated in Fig. 1. Generally, a 'replacement' DNA construct is made that carries two blocks with homology to the target, separated by a neomycin phosphotransferase II (neo) gene, which confers resistance to the drug G418. Electroporation is normally used to introduce the linearized construct into the ES cells, where it can undergo double homologous recombination, thereby replacing a portion of the target gene. The targeting efllciency is quite low, and so powerful selection methods are necessary to identify the ES cells that carry the desired modification. The first step is generally selection with G418, which removes all cells that failed to incorporate a n e o gene. Further selection is necessary, as most n e o genes will, unfortunately, insert in random genomic positions by a process of illegitimate recombination. One useful 'positive-negative' enrichment procedure devised by Mansour et al. [27] makes further use of a herpes virus thymidine .kinase gene placed outside the blocks of homology on the replacement construct. Homologous recombination will result in loss of the thymidine kinase gene, whereas illegitimate insertion will usually include the thymidlne kinase gene. The presence of the thymidine kinase gene can then be selected against by using anti-herpes virus drugs, such as gancyclovir. The next se-
lection step generally involves the polymerase chain reaction (PCR) using oligonucleotides that span the short block of homology. Only when the insertion is targeted, w i t h t h e n e o gene inserted next to this flanking sequence, will a clone score PCR-positive. After confirmation by Southern blot analysis, the targeted cells are injected into blastocysts which are implanted into surrogate mothers, where they give rise to chimeric mice that can be bred to give uniform heterozygotes. These can be used to make homozygotes carrying only modified versions of the gene. For a time there was concern that perhaps only those genes that are active in ES cells would have an appropriate open chromatin configuration to allow gene targeting. Johnson et al. [28..], however, have successfully targeted two genes, adipsin and adipocyte P2, that are adipose cell-specific and are not expressed at detectable levels in ES cells. Indeed, the targeting etticiencies were not even significantly reduced, suggesting that this technique would allow the alteration of any gene for which there is an available murine genomic clone for making the replacement construct. Another important technical question was answered by DeChiara et al. [29"] when they successfully used the 'positive-negative' selection scheme of Mansour et al. [27] to target disruption of the insulin-like growth factor (IGF)-II gene in ES cells and then proceeded to make germ-line chimeras. It had been suspected that gancyclovir treatment might reduce the totipotency of ES cells, but in the hands of DeChiara et al. there were no apparent ill effects. This renders the positive-negative selection procedure quite useful, as it gave an approximately tenfold enrichment for targeted ES cells. Interestingly, they also found that heterozygous mice, with one inactivated IGF-II gene, were dramatically smaller (about 60% body weight) than normal litter mates, further suggesting that IGF-U is an important growth factor during embryonic development. Homologous recombination in ES cells has also been used to study the developmental significance of the [32microglobulin gene, which is expressed during embryogenesis and has been suggested to have a number of nonimmunological functions. Mice with a homozygous disruption of this gene have now been produced and analyzed by two independent groups [30"°,31 "']. In each case, the mice were healthy, fertile and apparently normal except for the absence of CD4- 8 + T cells and little, if any, functional major histocompatibility complex class I antigens on cell surfaces. These results strongly suggest that development can proceed quite normally without 132microglobulin. Further characterization of these homozygous mice will be extremely useful in future studies focused on examining their altered immune systems. Moreover, this work is truly a landmark because it represents the first example in which mice were generated carrying a targeted modification in a gene for which there was no direct selection, thus establishing the general usefulness of the procedure. A number of genes, including homeobox genes and proto-oncogenes, for which an important role in devel-
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opment is suspected [27,28..-31 " , 3 2 , 3 3 " ] have now been targeted in ES cells. As homozygous mice carrying these and other targeted genes are made and analyzed, we should expect a wealth of information that will revolutionize the field of mammalian developmental genetics. Although these initial experiments were, in general, designed to inactivate endogenous genes totally, it is nevertheless clear that in time homologous recombination in ES cells will also be used to generate more subtle gene modifications to functionally dissect various coding domains. The work of Reid e t al. [ 3 4 " ] will allow the uge of 'in--out' procedures that can create targeted genes that are identical to their wild-type counterparts except for as little as a single base change. They have produced
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Fig. 1. Gene targeting. A replacement DNA construct is introduced into embryonal stem (ES) cells by electroporation. Homologous recombination can then insert part of this construct in place of a portion of the target gene. Selection procedures described in the text are used to identify targeted ES cells which are then injected into the host blastocysts. The blastocysts are implanted into the uteri of surrogate mothers where they give rise to chimeric mouse pups that carry a mixture of cells derived from the host blastocyst and the injected ES cells. The chimeras can be bred, and some of the progeny result from an ES cellderived germ cell. Some of these mice will be uniform heterozygotes, carrying one modified version of the target gene in every cell. Such heterozygotes can be interbred to give homozygotes. HSV-TK, herpes virus thymidine kinase; neo, gene encoding neomycin phosphotransferase II.
a hypoxanthine phosphoribosyltransferase (HPRT) gene construct that carries all of the appropriate control elements to allow high-level expression in ES cells. The great virtue of the HPRT gene is that it can be selected for both ways, for and against. This means that normal targeted gene disruptions in H P R T - ES cells can be made by simply using the HPRT gene in place of the n e o gene in a replacement vector. This disruption is then followed by the second stage of the alteration which is the 'otlt' step. A second replacement vector is used which carries the subtle modification to be introduced, and lacks any selectable marker gene. The appropriate double homologous recombination will insert the modification and simultaneously delete the HPRT gene from the target lo-
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Mammaliangene studies cus. The altered ES cells are highly enriched following growth in 6-thioguanine (which selects against the presence of HPRT) because random insertions of the second replacement vector do not delete the HPRT gene. The net result is a target gene with the desired base changes, but carrying no selectable marker gene that might itself influence gene function.
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BALLINGR, MUTTERG, GRUSS P, KESSELM: Craniofacial abnormalities i n d u c e d by ectopic expression of t h e h o m e o b o x g e n e Hox 1.1 in transgenic mice. Cell 1989, 58:337-347. Very nice study that shows abnormal development resulting from ectopic Hox 1.1 expression. 9.
KESSEL M, BALLING R, GRUSS P: Variations of cervical vertebrae
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after expression of a Hox 1.1 transgene in mice. Cell 1990, 61:301-308. Further analysis of [Lactin-Hox 1.1 transgenics shows abnormal development of cervical somites. 10.
Future prospects There are now some very powerful techniques for manipulating the murine genome. New genetic information can be quickly and efficiently added by pronuclear microinjection, and targeted gene knock-outs or subtle modifications can be accomplished by homologous recombination in ES cells. In each case, mice are produced that have an altered genetic content in all their cells. These techniques are proving useful in the functional analysis of mammalian genes, providing new insight into the genetic basis of a variety of biological processes. Likewise, the long-term practical consequences are sure to be significant as this ability to modify the genome is gradually extended to other species, including Homo sapiens.
References and recommended reading • ••
Of interest Of outstanding interest
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BEHRINGERRR, RYAN TM, REILLY MP, ASAKURAT, PALMITER RD, BRINSTER RL, TOWNES T: Synthesis of functional h u m a n hemoglobin in transgenic mice. Science 1989, 245:971-973. Transgenic mice carrying both h u m a n 0c and ~3-globin genes with appropriate control elements are shown to synthesize normal h u m a n hemoglobin. •,
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GREAVESDR, FRASER P, VIDAL /VIA, HEDGES MJ, ROPERS D, LUZZATrO L, GROSVELD F: A transgenic m o u s e m o d e l of sickle cell disorder. Nature 1990, 343:183-185. Transgenic mice making h u m a n sickle cell [3-globin and h u m a n ~globin are found to have red blood cells that readily sickle upon deoxygenation. Some in vivo sickling was also observed. • •
12. ••
HECKELJL, SANDGRENEP, DEGEN JL, PAKMrI~RRD, BRINSTERRL: Neonatal bleeding in transgenic mice expressing urokinasetype plasminogen activator. Cell 1990, 62:447~456. Transgenic mice with the albumin enhancer and promoter driving expression of the urokinase plasminogen activator are found to suffer spontaneous intestinal and intra-abdominal bleeding. 13. ••
ScoTt M, FOSTER D, MIRENDAC, SERBAND, CONFALF, WALCHLI M, TORCHIAM, GROTH D, CARLSONG, DEARMONDSJ, WESTAWAY D, PRUSINER SB: Transgenic mice expressing h a m s t e r prion protein p r o d u c e species-specific scrapie infectivity and amyloid plaques. Cell 1989, 59:847-857. Transgenic mice producing hamster prion protein are found to develop a hamster-type scmpie after innoculation with hamster prions. Non-transgenic mice fail to develop scrapie in similar circumstances. 14.
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ScoTr MP, TAMKUMJ'Vd, HARTZELLGW m: The structure and function of t h e h o m e o d o m a i n . Biochem Biophys Acta 1989, 989:25-48. A good review of h o m e o b o x genes. 6.
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WOLGEMUTHDJ, BEHRINGER RR, MOSTOLLERMP, BRINSTER RL, PALMITER RD: Transgenic mice overexpressing t h e m o u s e h o m e o b o x - c o n t a i n i n g g e n e H o x 1.4 exhibit abnormal gut development. Nature 1989, 337:464-467.
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ZELLERR, JACKSON-GRUSBY L, LEDER P: The lilTlb deformity g e n e is required for apical ectodermal ridge differentiation and anteroposterior limb pattern formation. Genes Dev 1989, 3:1481-1492. Embryologic characterization of the limb deformity phenotype. 16.
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MCNEISHJD, THAYERJ, WAHaNG K, SUUKKK, POTTER SS, SCOTT WJ: Phenotypic characterization of t h e transgenic m o u s e insertional mutation, legless. J Exp Zool 1990, 253:151-162. A more detailed analysis of the phenotype of newborn legless mice than appeared in the original Science report [16]. Includes a description of the situ inversus aspect. KRULEWSKITF, NEUMANN PE, GORDON J~X[: Insertional m u t a tion in a transgenic m o u s e allelic w i t h Purkinje cell degeneration. Proc Natl Acad Sci USA 1989, 86:3709-3712. A description of an insertional mutation in the pcdlocus. Homozygotes have a total absence of Purkinje cells, and other interesting abnormalities. 18. • •
19.
GORDONfW, UEHHNGERJ, DAYANI N, TALANSKYBE, GORDON
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M, RUDOMEN GS, NEUMANNPE: Analysis of t h e hotfoot (ho)
locus by creation o f an insertional m u t a t i o n in a transgenic mouse. Dev Biol 1990, 137:349-358. Description of an insertional mutation that is allelic with the ho locus. Another interesting case where neurologic and sperrnatogenesis defects are coupled. 20. •
MACGREGORGR, RUSSELLLD, VAN BECK MEAB, HANTEN GR, KOVAC MJ, KOZAKCA, MEISTRICHML, OVERBEEKPA: Symplastic
T r a n s g e n i c a n i m a l s Potter
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JOHNSON RS, SHENG M, GREENBERG ME, KOLODNER RD, PhPA1OANNOUVE, SPIEGELMANBM: Targeting of n o n e x p r e s s e d
g e n e s in embryonic stem cells via h o m o l o g o u s recombination. Science 1989, 245:1234-1236. Genes that are not expressed in ES cells were shown to target with high elticiency. 29. .,
DECHIARATM, EFSTRATIADISA, ROBERTSONEJ: A growth-deftciency p h e n o t y p e in heterozygous mice carrying an insulinlike g r o w t h factor II g e n e disrupted by targeting. N a t u r e 1990, 345:784~0. The 'positive-negative' enrichment scheme was shown to give tenfold enrichment without harming ES cell totipotency. 30. ••
ZIJLSTRAM, BIX M, SIM~STER NE, LOVING JM, RAULET DH, JAENISCHR: ~2-microglobulin deficient mice lack C D 4 - 8 + cytolytic T cells. N a t u r e 1990, 344:742-746. Successful targeting of a gene that is not directly selectable, and generation of homozygous mice. Description of resulting phenotype. KOLLERBH, MARRACKP, KAPPLERJ'~V, SMITHIES O: Normal dev e l o p m e n t o f mice deficient in ~2M, Class I proteins and CD8 + T cells. Science 1990, 248:1227-1230. As in [30oo], successful knock-out of 132-microglobulin gene and generation of homozygous mice. 31. ..
32.
JOYNERA, SKARNESWC, ROSSANTJ: Production of a Nmutation in m o u s e En-2 gene by h o m o l o g o u s recombination in embryonic s t e m cells. N a t u r e 1989, 338:153-155.
33. °°
SCHWARTZBERGPL, ROBERTSON EJ, GOFF SP: Targeted gene disruption of t h e e n d o g e n o u s c-abl locus by homologous recombination w i t h DNA e n c o d i n g a selectable fusion protein. P r o c N a t l A c a d Sci USA 1990, 87:3210-3214. Describes the use of a promoterless n e o fusion construct to target the c-abl locus.
34. °°
REID LH, GREGG RG, SMITHIES O, KOLLERBH: Regulatory elem e n t s in the introns of t h e h u m a n HPRT g e n e are necessary for its expression in embryonic s t e m ceils. Proc N a t l : A c a d Sci USA 1990, 87:4299-4303. This work shows that some intronic sequences are necessary for high level HPRT expression in ES cells. The resulting HPRT construct should be extremely useful in generating subtle changes in targeted genes.
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Introduction Because of the pioneering efforts of a number of investigators, it is now possible to manipulate the mammalian genome by a powerful array of methods. The ~¢orkhorse procedure of the past decade has been the pronuclear microinjection technique, first published by Gordon et al. [1]. This approach allows one to add genes to the genome efficiently, producing a mouse, for example, that carries new genetic information in every cell of its body. Pronuclear microinjection has been, and will continue to be, useful for a wide variety of experiments. Another technique uses homologous recombination in embryonal stem (ES) cells to produce mice with endogenous genes modified in a very precise manner. This ability to target changes in specific genes promises to be an important tool in the functional analysis of the mammalian genome. The biological impact of genetic alterations can be observed directly in the phenotypes of the transgenic animals, thereby yielding insight into a number of biological processes. Transgenic studies of carcinogenesis and the immune system have been reviewed recently [2 o]. Here, I will focus primarily on the advances made in the last year or so in using transgenic animals to study mammalian development.
Pronuclear microinjection Mammalian genome manipulation techniques generally involve the initial modification of a single cell which is then used to produce a mouse. For the pronuclear microinjection method, this cell is the zygote, or fertilized egg. The technique is conceptually straightforward, although the underlying molecular mechanisms of transgene insertion remain poorly understood. A few hundred copies of the gene construct to be added to the genome are simply microinjected into the male (larger) pronucleus a few hours before its fusion with the female pronucleus. Remarkably, in a significant fraction of these onecell embryos, some of the added DNA will covalently integrate into a single, random genomic position by a process of illegitimate (non-homologous) recombination. The in-
serted foreign DNA is referred to as a transgene, and it generally consists of a head-to-tail concatemer of a variable number of injected molecules. The injected cells are then simply implanted into the oviducts of surrogate mothers and 3 weeks later mouse pups are born. Generally at 2-3 weeks of age, their DNA is analyzed to determine which of them are actually transgenic (usually about 10-30%). Transgenic mice were originally used almost exclusively for gene expression studies [3]. In many respects, they represent an ideal in vivo expression system for the analysis of ctX-acting controlling elements associated with genes. DNA constructs lacMng putative control elements, or carrying elements connected to a reporter gene, can be tested for activity in these mice, where all cell types and all developmental times are available for analysis. Despite the disadvantages of cost, technical difficulty, and the variability of expression pattems resulting from different integration sites, this approach nevertheless remains powerful and is still often used. This is because, for many genes, an appropriately expressing cell type is not readily grown in culture, and in any event it is clear that cell culture gene assay systems do not recognize all of the control elements necessary for appropriate in vivo expression [4]. These transgenic mice are remarkably unperturbed by the presence within their cells of a promoter that is often driving high-level production of some bacterial reporter gene-encoded protein. The completely normal appearance of these mice suggests a considerable vigor within the system which can readily tolerate significant loads of innocuous foreign protein. With care, however, it is possible to design transgenes that are biologically active and alter the phenotype of the mouse in an informative way.
Dominant homeobox transgenes The homeobox genes were originally found in Drosop hila, where it was observed that a number of developmentally important genes carry a highly conserved 180bp sequence encoding a DNA-binding domain [5 °]. These genes have an important role in controlling
Abbreviations ES~embryonal stem; ho---hotfoot; HPRT--hypoxanthine phosphoribosyltransferase;IGF~insulin-like growth factor; Igl--legless; Id~limb deformity; PCR--polymerasechain reation. (~) Current Biology Ltd ISSN 0958-1669
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Mammaliangene studies Drosophila development by regulating the expression of other h o m e o b o x genes as well as 'subservient' batteries of genes. Some of the homeobox genes help to establish the pattern of segmentation; others determine segment identity. Some of these genes are apparently master switches that control the developmental destinies of groups of cells. An Antennapedia mutation, for example, can cause legs to form where the antennae would normaUy be. The homeobox is so evolutionarily well conserved that it has been possible to retrieve over 35 genes with extensive homology from the murine genome, some of which have now been studied in detail. Expression patterns of murine homeobox genes during development have been determined primarily by in situ hybridization to non-transgenic embryos, although the use of [~-galactosidase reporter transgenes is becoming increasingly popular, for example in localizing DNA control elements that drive region-specific expression [6.]. The results of these investigations are extremely interesting, and certainly suggest that the murine homeobox genes have a role in development; nevertheless, the evidence remains circumstantial. Without any known mutations in these genes, their true biological functions remain uncertain. Wolgemuth et al. [7] were the first to functionally analyze the murine h o m e o b o x genes (Iqox genes) by generating transgenic mice; they used a DNA construct that included the H o x l . 4 gene with 10kb of upstream DNA and an SV40 sequence tag at the 3' end to distinguish transcripts from those of the endogenous gene. In situ hybridization revealed that the transgenic mice, which had extra copies of Hoxl.4, expressed higher levels of transcripts, particularly in the gut. Interestingly, this resulted in a developmental abnormality, termed megacolon, in which the bowel is grossly distended and feces are not easily extruded from the distal end of the colon. These experiments were the first to demonstrate a developmental malformation resulting from inappropriate expression of a mammalian h o m e o b o x gene. Bailing et al. [8".] carried this approach one step further when they made transgenic mice with the chicken [3-actin promoter connected to the coding sequence of Hoxl. 1. This heterologous promoter produced almost ubiquitous low-level expression of the Hoxl.1. gene, allowing a variety of tissues to be tested for a possible developmental response. The resulting mice were indeed malformed, with craniofacial abnormalities including open eyes at birth, detached pinnae and cleft secondary palate. The mice invariably died within 14 days of birth. Careful analysis of the more severely affected mice also revealed some intriguing abnormalities in the development of the cervical somites [9"'].
Dominant transgenes and human disease A number of extremely interesting dominant transgenes that promise to increase our understanding of certain human diseases deserve a mention. For example, it is
now possible to generate mice that produce human hemoglobin [10-.,11 ..], and with further refinements it appears likely that an excellent animal model for sickle cell disease will result. In another study, Heckel el al. [12 ..] have produced mice that overexpress urokinasetype plasminogen activator, and consequently suffer a severe bleeding disorder. Finally, Scott et al. [13"] have demonstrated the importance of prion protein in determining scrapie susceptibility, incubation time and neuropathology by producing transgenic mice that express the hamster prion gene.
Insertional mutations A useful by-product of the promoter assay and other types of transgenic mouse experiments described above is the generation of insertional mutations in endogenous genes. That is, in a small fraction (about 5%) of transgenic lines, the transgene will insert by chance into a chromosomal position that results in the inactivation of the gene originally residing there. Such mutations are particularly useful because of the molecular handle provided by the known transgene sequences, which allows molecular retrieval and analysis of the disrupted gene. Furthermore, the genes identified by insertional mutagenesis are known to be interesting from the outset, as one begins with a mutant mouse displaying a phenotype resulting from the inactivation of the gene. A number of interesting insertional mutations have recently been better described than before or reported for the first time. The limb deformffy (ld) mutation, first characterized by Woychik et al. [14], is certainly among the most developmentally fascinating. They found that mice homozygous for the transgene insertion displayed a phenotype that included synostoses (fusion) of the long bones of both forelimbs and hindlimbs, oligodactyly and syndactyly of the hand and foot bones, and a high frequency of unilateral and bilateral renal aplasias. This mutation was shown to be aUelic to two previously found spontaneous ld alleles. In a careful embryologic analysis of ld/ldmutants, Zeller et al. [15 " ] have found more recently that limb buds appear defective as early as day 10, with a shortened anterior-posterior axis and an apical ectodermal ridge that never achieves the correct morphology. At later times in development, it was shown that the ulna and radius form as separate elements that then fuse abnormally, whereas the tibia and fibula actually form as a single unit. Moreover, molecular analysis of this locus is now revealing an intriguing story of a very large gene that gives rise to several distinct transcripts during development. The insertional mutation named legless (lgl) also affects limb development [16]. The mutant mice (lgFlgi) are born with no hindlimb structure distal to the femur. Furthermore, they lack anterior distal portions of the forelimbs and anterior portions of the brain. In a more detailed description of the newborn mutant phenotype, McNeish et al. [17.] mention another interesting char-
Transgenic animals Potter acteristic. They observed that 50% have situs inversus, (with inverted viscera). Chromosome mapping results and complementation tests strongly suggest that this insertional mutation is allelic with a classic murine mutation locus. Mouse DNAs flanking the transgene insertion have been recovered and their molecular analysis is underway (Singh, Supp, McNeish, Copeland, Jenkins and Potter, unpublished data). Krulewski etal. [18..] have described an insertional mutation in which homozygotes have a number of fascinating neurologic defects, including a total absence of Purkinje cells, degeneration of photoreceptor ceils from the retina, and a loss of almost all mitral cells from the olfactory bulb. Most of the homozygote males (18 out of 22) were also sterile, with histologic analysis revealing no sperm in the epididymis. This insertional mutation was shown to be allelic with the previously described classic mutant locus, pcd ( Purkinje cell degeneration), which exhibits a similar phenotype. Molecular analysis of this locus is in the initial stages, with mouse DNA apparently flanking the transgene insertion retrieved, but with no coding sequences yet identified. Remarkably, Gordon's group has also identified another insertional mutation that affects both neurologic development and spermatogenesis [19"]. The homozygotes show an ataxic gait when about 2 weeks old, the severity of which increases with age. Histologic analysis revealed no detectable neurologic abnormalities. The homozygous mice were functionally sterile and, although sperm counts were normal, the mutant males were shown to produce sperm defective in their ability to penetrate the egg zona. Interestingly, this insertional mutation, too, was found to be allelic with a previously identified 'classic' mutation in the mouse genome termed ho~oot (ho). Once again, flanking sequences abutting the transgene insert have been retrieved and molecular analysis has begun. Another insertional mutation resulting in male sterility has been described by MacGregor et al. [20.]. In this case, the homozygous mice are apparently normal in all other respects, although developing spermatids do not mature properly. This mutation, named sys, is not allelic with any previously characterized locus. Xiang etal. [21 ..] have described an insertional mutation resulting in mice that are much reduced in size. Matings showed that this 'mini-mouse' insertional mutation is allelic with the spontaneous mutation pygmy. These mice are proportionately correct, except for short ears, and have normal circulating levels of growth hormone and somatostatin. It is to be hoped that molecular analysis of the disrupted gene will lead to a better understanding of comparable human dwarf syndromes. It is clear that the careful screening of transgenic lines for the presence of possible recessive insertional mutations provides a powerful method for randomly scanning the murine genome for genes of particular interest. Mthough this approach is certainly yielding dividends, there are often serious ditficulties. First, in some cases the transgene is highly methylated and as a result is very refractory to cloning in almost all Escherichia coli strains, which
are capable of recognizing foreign methylation patterns and selectively destroying DNA. Cells that lack both the McrA and McrB systems still restrict some transgenes, but as new cells are now available that are more completely restriction-deficient (DIt10B), this problem is disappearing. A second and more common difficulty results from the complexity of the transgene insertion. It is often, for example, found associated with a deletion of genomic DNA at the insertion site, which can range in size from very small (less than 1 kb), to very large (more than 100 kb). Extensive deletions obviously mean that a large region must be scrutinized for the presence of the gene of interest. Perhaps even more perplexing, however, are the cases where the transgene is actually mixed with murine genomic DNA 'islands'. These islands, which can also vary in size, greatly complicate the analysis. It is clear that the degree of chromosomal destruction rendered by the transgene insertion must be carefully assessed, so that all possible candidate genes can be evaluated. On the positive side, however, the molecular tag of the transgene insertion will almost always allow the investigator to retrieve a mouse genomic sequence that is at least 'close' (estimated as within 100kb) to the dismpted gene. Although this will still leave much work to be done, it nevertheless represents an excellent starting point, at least by the standards of those who do 'reverse genetics' or cloning based on chromosomal position. Also, it is becoming clear that a surprisingly large fraction of insertional mutations are allelic with previously described 'classic' mutations. This can be extremely useful in the identification of the gene of interest, as one can compare wild-type, classic mutant and insertional mutant transcripts. The final diagnostic tests, however, are still rescue of the wild-type phenotype by adding back a wildtype copy of the gene into transgenic mice, or knock-out of the gene in wild-type mice, using ES cells, to generate the phenotype independently.
Enhancer traps, gene traps, and retroviral insertions The enhancer trap approach [22,23..] has both advantages and disadvantages when compared with the standard insertional mutagenesis described above. This technique uses a ]3-galactosidase gene connected to a weak promoter as a transgene. On occasion, the expression patterns of such a transgene which are readily detected by Xgal (5-bromo-4-chloro-3-indoyl-B-D-galactopyranoside) staining, are particularly interesting. So with this approach one begins the molecular analysis knowing something of the expression pattern, but lacking a phenotype. It must be noted that interesting expression patterns are not always associated with interesting gene function. Moreover, the molecular search for the gene is further complicated by the ability of enhancers to work from considerable distances, thus expanding the genornic region that must be analyzed.
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Mammalian gene studies The gene trap method in some ways combines the best of the enhancer trap and standard insertional mutagenesis techniques. In this method, a promoterless, but spliceacceptor-pre-fixed [3-galactosidase gene is used, and insertion into an endogenous gene can result in expression. Hence, one begins the molecular analysis with knowledge of the expression pattern, an excellent molecular handle, and probably a functionally disrupted gene. Insertional mutations can also be generated by retroviral infection of mouse embryos or ES cells [24,25]. In this method, one copy of the retroviral genome integrates 'cleanly', with no deletion or scrambling of host genomic DNA. This approach has been used effectively in the past and still holds great promise for the future. The very real advantage it offers is the relative ease of molecular characterization of the insertional mutations.
Gene targeting Although the pronuclear microinjection procedure is quite effective for adding DNA concatemers to random genomic locations, there are many problems that are better approached by targeted modification of endogenous genes. For example, in the cases of the many cloned genes for which there are no known classic mutations, inactivation and/or modification of endogenous copies of these genes would greatly facilitate a functional analysis. This can now be achieved by homologous recombination in ES cells. ES cells are derived from the inner cell mass of a blastocyst, and can be cultured for prolonged periods and yet retain totipotency [26]. That is, when reintroduced into a blastocyst they can contribute to all cell lineages, including the germ line, of the resulting embryo. The general scheme for targeted gene modification of ES cells is illustrated in Fig. 1. Generally, a 'replacement' DNA construct is made that carries two blocks with homology to the target, separated by a neomycin phosphotransferase II (neo) gene, which confers resistance to the drug G418. Electroporation is normally used to introduce the linearized construct into the ES cells, where it can undergo double homologous recombination, thereby replacing a portion of the target gene. The targeting efllciency is quite low, and so powerful selection methods are necessary to identify the ES cells that carry the desired modification. The first step is generally selection with G418, which removes all cells that failed to incorporate a n e o gene. Further selection is necessary, as most n e o genes will, unfortunately, insert in random genomic positions by a process of illegitimate recombination. One useful 'positive-negative' enrichment procedure devised by Mansour et al. [27] makes further use of a herpes virus thymidine .kinase gene placed outside the blocks of homology on the replacement construct. Homologous recombination will result in loss of the thymidine kinase gene, whereas illegitimate insertion will usually include the thymidlne kinase gene. The presence of the thymidine kinase gene can then be selected against by using anti-herpes virus drugs, such as gancyclovir. The next se-
lection step generally involves the polymerase chain reaction (PCR) using oligonucleotides that span the short block of homology. Only when the insertion is targeted, w i t h t h e n e o gene inserted next to this flanking sequence, will a clone score PCR-positive. After confirmation by Southern blot analysis, the targeted cells are injected into blastocysts which are implanted into surrogate mothers, where they give rise to chimeric mice that can be bred to give uniform heterozygotes. These can be used to make homozygotes carrying only modified versions of the gene. For a time there was concern that perhaps only those genes that are active in ES cells would have an appropriate open chromatin configuration to allow gene targeting. Johnson et al. [28..], however, have successfully targeted two genes, adipsin and adipocyte P2, that are adipose cell-specific and are not expressed at detectable levels in ES cells. Indeed, the targeting etticiencies were not even significantly reduced, suggesting that this technique would allow the alteration of any gene for which there is an available murine genomic clone for making the replacement construct. Another important technical question was answered by DeChiara et al. [29"] when they successfully used the 'positive-negative' selection scheme of Mansour et al. [27] to target disruption of the insulin-like growth factor (IGF)-II gene in ES cells and then proceeded to make germ-line chimeras. It had been suspected that gancyclovir treatment might reduce the totipotency of ES cells, but in the hands of DeChiara et al. there were no apparent ill effects. This renders the positive-negative selection procedure quite useful, as it gave an approximately tenfold enrichment for targeted ES cells. Interestingly, they also found that heterozygous mice, with one inactivated IGF-II gene, were dramatically smaller (about 60% body weight) than normal litter mates, further suggesting that IGF-U is an important growth factor during embryonic development. Homologous recombination in ES cells has also been used to study the developmental significance of the [32microglobulin gene, which is expressed during embryogenesis and has been suggested to have a number of nonimmunological functions. Mice with a homozygous disruption of this gene have now been produced and analyzed by two independent groups [30"°,31 "']. In each case, the mice were healthy, fertile and apparently normal except for the absence of CD4- 8 + T cells and little, if any, functional major histocompatibility complex class I antigens on cell surfaces. These results strongly suggest that development can proceed quite normally without 132microglobulin. Further characterization of these homozygous mice will be extremely useful in future studies focused on examining their altered immune systems. Moreover, this work is truly a landmark because it represents the first example in which mice were generated carrying a targeted modification in a gene for which there was no direct selection, thus establishing the general usefulness of the procedure. A number of genes, including homeobox genes and proto-oncogenes, for which an important role in devel-
Transgenic animals Potter
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opment is suspected [27,28..-31 " , 3 2 , 3 3 " ] have now been targeted in ES cells. As homozygous mice carrying these and other targeted genes are made and analyzed, we should expect a wealth of information that will revolutionize the field of mammalian developmental genetics. Although these initial experiments were, in general, designed to inactivate endogenous genes totally, it is nevertheless clear that in time homologous recombination in ES cells will also be used to generate more subtle gene modifications to functionally dissect various coding domains. The work of Reid e t al. [ 3 4 " ] will allow the uge of 'in--out' procedures that can create targeted genes that are identical to their wild-type counterparts except for as little as a single base change. They have produced
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Fig. 1. Gene targeting. A replacement DNA construct is introduced into embryonal stem (ES) cells by electroporation. Homologous recombination can then insert part of this construct in place of a portion of the target gene. Selection procedures described in the text are used to identify targeted ES cells which are then injected into the host blastocysts. The blastocysts are implanted into the uteri of surrogate mothers where they give rise to chimeric mouse pups that carry a mixture of cells derived from the host blastocyst and the injected ES cells. The chimeras can be bred, and some of the progeny result from an ES cellderived germ cell. Some of these mice will be uniform heterozygotes, carrying one modified version of the target gene in every cell. Such heterozygotes can be interbred to give homozygotes. HSV-TK, herpes virus thymidine kinase; neo, gene encoding neomycin phosphotransferase II.
a hypoxanthine phosphoribosyltransferase (HPRT) gene construct that carries all of the appropriate control elements to allow high-level expression in ES cells. The great virtue of the HPRT gene is that it can be selected for both ways, for and against. This means that normal targeted gene disruptions in H P R T - ES cells can be made by simply using the HPRT gene in place of the n e o gene in a replacement vector. This disruption is then followed by the second stage of the alteration which is the 'otlt' step. A second replacement vector is used which carries the subtle modification to be introduced, and lacks any selectable marker gene. The appropriate double homologous recombination will insert the modification and simultaneously delete the HPRT gene from the target lo-
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Mammaliangene studies cus. The altered ES cells are highly enriched following growth in 6-thioguanine (which selects against the presence of HPRT) because random insertions of the second replacement vector do not delete the HPRT gene. The net result is a target gene with the desired base changes, but carrying no selectable marker gene that might itself influence gene function.
8. ••
BALLINGR, MUTTERG, GRUSS P, KESSELM: Craniofacial abnormalities i n d u c e d by ectopic expression of t h e h o m e o b o x g e n e Hox 1.1 in transgenic mice. Cell 1989, 58:337-347. Very nice study that shows abnormal development resulting from ectopic Hox 1.1 expression. 9.
KESSEL M, BALLING R, GRUSS P: Variations of cervical vertebrae
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after expression of a Hox 1.1 transgene in mice. Cell 1990, 61:301-308. Further analysis of [Lactin-Hox 1.1 transgenics shows abnormal development of cervical somites. 10.
Future prospects There are now some very powerful techniques for manipulating the murine genome. New genetic information can be quickly and efficiently added by pronuclear microinjection, and targeted gene knock-outs or subtle modifications can be accomplished by homologous recombination in ES cells. In each case, mice are produced that have an altered genetic content in all their cells. These techniques are proving useful in the functional analysis of mammalian genes, providing new insight into the genetic basis of a variety of biological processes. Likewise, the long-term practical consequences are sure to be significant as this ability to modify the genome is gradually extended to other species, including Homo sapiens.
References and recommended reading • ••
Of interest Of outstanding interest
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BEHRINGERRR, RYAN TM, REILLY MP, ASAKURAT, PALMITER RD, BRINSTER RL, TOWNES T: Synthesis of functional h u m a n hemoglobin in transgenic mice. Science 1989, 245:971-973. Transgenic mice carrying both h u m a n 0c and ~3-globin genes with appropriate control elements are shown to synthesize normal h u m a n hemoglobin. •,
11.
GREAVESDR, FRASER P, VIDAL /VIA, HEDGES MJ, ROPERS D, LUZZATrO L, GROSVELD F: A transgenic m o u s e m o d e l of sickle cell disorder. Nature 1990, 343:183-185. Transgenic mice making h u m a n sickle cell [3-globin and h u m a n ~globin are found to have red blood cells that readily sickle upon deoxygenation. Some in vivo sickling was also observed. • •
12. ••
HECKELJL, SANDGRENEP, DEGEN JL, PAKMrI~RRD, BRINSTERRL: Neonatal bleeding in transgenic mice expressing urokinasetype plasminogen activator. Cell 1990, 62:447~456. Transgenic mice with the albumin enhancer and promoter driving expression of the urokinase plasminogen activator are found to suffer spontaneous intestinal and intra-abdominal bleeding. 13. ••
ScoTt M, FOSTER D, MIRENDAC, SERBAND, CONFALF, WALCHLI M, TORCHIAM, GROTH D, CARLSONG, DEARMONDSJ, WESTAWAY D, PRUSINER SB: Transgenic mice expressing h a m s t e r prion protein p r o d u c e species-specific scrapie infectivity and amyloid plaques. Cell 1989, 59:847-857. Transgenic mice producing hamster prion protein are found to develop a hamster-type scmpie after innoculation with hamster prions. Non-transgenic mice fail to develop scrapie in similar circumstances. 14.
HANAHAN D: Transgenic mice as probes into c o m p l e x sys• terns. Science 1989, 246:1265-1275. A review primarily of pronuclear microinjection transgenic m o u s e studies of the immune system and carcinogenesis. 2.
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BRINSTERRL, PAHVI1TERRD: Introduction of g e n e s into t h e g e r m lines of animals. The Harvey Lectures 1986, series 80:1~8. BRINSTERRL, ALLENJM, BEHRINGER RR, GELINAS RE, PALMITER RD: Introns increased transciptional efficiency in transgenic mice. Proc Natl Acad Sci USA 1988, 85:836q~0.
5. •
ScoTr MP, TAMKUMJ'Vd, HARTZELLGW m: The structure and function of t h e h o m e o d o m a i n . Biochem Biophys Acta 1989, 989:25-48. A good review of h o m e o b o x genes. 6.
TUGGLECK, ZAKANYJ, CIANETrl L, PESCHLEC, NGUYEN-HUUMC: Region-specific e n h a n c e r s near two mammalian h o m e o b o x g e n e s d e f i n e a d j a c e n t rostrocaudal domains in t h e central nervous system. Genes Dev 1990, 4:180-189. The authors describe ~ e use of a ]3-galactosidase reporter gene to identify regulatory elements of the H o x l . 3 and Hox5.1 genes that drive region-specific expression. •
7.
WOLGEMUTHDJ, BEHRINGER RR, MOSTOLLERMP, BRINSTER RL, PALMITER RD: Transgenic mice overexpressing t h e m o u s e h o m e o b o x - c o n t a i n i n g g e n e H o x 1.4 exhibit abnormal gut development. Nature 1989, 337:464-467.
WOYCHIKRP, STEWARTTA, DAVIS LG, D'EUSTACHIO P, LEDERP: An inherited limb deformity created by insertioual mutagenesis in a transgenic mouse. Nature 1985, 318:36-40.
15. ••
ZELLERR, JACKSON-GRUSBY L, LEDER P: The lilTlb deformity g e n e is required for apical ectodermal ridge differentiation and anteroposterior limb pattern formation. Genes Dev 1989, 3:1481-1492. Embryologic characterization of the limb deformity phenotype. 16.
MCNEISH J, ScoTt W, POTTER SS: Legless, a novel mutation found in PH 51-1 transgenic mice. Science 1988, 241:837-839.
17. •
MCNEISHJD, THAYERJ, WAHaNG K, SUUKKK, POTTER SS, SCOTT WJ: Phenotypic characterization of t h e transgenic m o u s e insertional mutation, legless. J Exp Zool 1990, 253:151-162. A more detailed analysis of the phenotype of newborn legless mice than appeared in the original Science report [16]. Includes a description of the situ inversus aspect. KRULEWSKITF, NEUMANN PE, GORDON J~X[: Insertional m u t a tion in a transgenic m o u s e allelic w i t h Purkinje cell degeneration. Proc Natl Acad Sci USA 1989, 86:3709-3712. A description of an insertional mutation in the pcdlocus. Homozygotes have a total absence of Purkinje cells, and other interesting abnormalities. 18. • •
19.
GORDONfW, UEHHNGERJ, DAYANI N, TALANSKYBE, GORDON
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M, RUDOMEN GS, NEUMANNPE: Analysis of t h e hotfoot (ho)
locus by creation o f an insertional m u t a t i o n in a transgenic mouse. Dev Biol 1990, 137:349-358. Description of an insertional mutation that is allelic with the ho locus. Another interesting case where neurologic and sperrnatogenesis defects are coupled. 20. •
MACGREGORGR, RUSSELLLD, VAN BECK MEAB, HANTEN GR, KOVAC MJ, KOZAKCA, MEISTRICHML, OVERBEEKPA: Symplastic
T r a n s g e n i c a n i m a l s Potter
spermatids (sys): A recessive insertional mutation in mice causing a defect in spermatogenesis. Proc N a t l A c a d Sci USA 1990, 87:5016-5020. An insertional mutation resulting in defective spermatogenesis, but no other detectable problems. Apparently not allelic with any previous locus. 21. •°
XIANGX, BENSON KF, CHADA K: Mini mouse: disruption of the p y g m y locus in a transgenic insertional mutant. Science 1990, 24:967-969. Description of an insertional mutation in the p y g m y locus that results in dwarfism, 22.
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23. •°
GOSSLERA, JOYIVERA, ROSSANTJ, SKARNESW: Mouse embryonic s t e m cells a n d reporter constructs to d e t e c t developmentally regulated genes. Science 1989, 244:463-465. Describes the use of ES cells for enhancer trap and gene trap experiments w i t h / a c Z c o n s t m c t s . 24.
JAENISCHR, HARBERSK, SCHNIEKE A, LOHLER J, CHUMAKOV I, JAHNER D, GROTKOPP D, HOFFMAN E: Germline integration of Maloney leukemia virus at t h e Mov 13 locus leads to recessive lethal mutation and early embryonic death. Cell 1983, 32:209-216.
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BRADLEYA, EVANS M, KAUFMAN MH, ROBERTSON E: Formation o f germ-line chimeras from embryo-derived teratocarcinoma cell lines. N a t u r e 1984, 309:255-256.
27.
MANSOURSL, THOMAS KR, CAPECCHI MR: Disruption of t h e p r o t o - o n c o g e n e int-2 in m o u s e embryo-derived s t e m cells: a general strategy for targeting mutations to non-selectable genes. N a t u r e 1988, 336:348-352.
28. °°
JOHNSON RS, SHENG M, GREENBERG ME, KOLODNER RD, PhPA1OANNOUVE, SPIEGELMANBM: Targeting of n o n e x p r e s s e d
g e n e s in embryonic stem cells via h o m o l o g o u s recombination. Science 1989, 245:1234-1236. Genes that are not expressed in ES cells were shown to target with high elticiency. 29. .,
DECHIARATM, EFSTRATIADISA, ROBERTSONEJ: A growth-deftciency p h e n o t y p e in heterozygous mice carrying an insulinlike g r o w t h factor II g e n e disrupted by targeting. N a t u r e 1990, 345:784~0. The 'positive-negative' enrichment scheme was shown to give tenfold enrichment without harming ES cell totipotency. 30. ••
ZIJLSTRAM, BIX M, SIM~STER NE, LOVING JM, RAULET DH, JAENISCHR: ~2-microglobulin deficient mice lack C D 4 - 8 + cytolytic T cells. N a t u r e 1990, 344:742-746. Successful targeting of a gene that is not directly selectable, and generation of homozygous mice. Description of resulting phenotype. KOLLERBH, MARRACKP, KAPPLERJ'~V, SMITHIES O: Normal dev e l o p m e n t o f mice deficient in ~2M, Class I proteins and CD8 + T cells. Science 1990, 248:1227-1230. As in [30oo], successful knock-out of 132-microglobulin gene and generation of homozygous mice. 31. ..
32.
JOYNERA, SKARNESWC, ROSSANTJ: Production of a Nmutation in m o u s e En-2 gene by h o m o l o g o u s recombination in embryonic s t e m cells. N a t u r e 1989, 338:153-155.
33. °°
SCHWARTZBERGPL, ROBERTSON EJ, GOFF SP: Targeted gene disruption of t h e e n d o g e n o u s c-abl locus by homologous recombination w i t h DNA e n c o d i n g a selectable fusion protein. P r o c N a t l A c a d Sci USA 1990, 87:3210-3214. Describes the use of a promoterless n e o fusion construct to target the c-abl locus.
34. °°
REID LH, GREGG RG, SMITHIES O, KOLLERBH: Regulatory elem e n t s in the introns of t h e h u m a n HPRT g e n e are necessary for its expression in embryonic s t e m ceils. Proc N a t l : A c a d Sci USA 1990, 87:4299-4303. This work shows that some intronic sequences are necessary for high level HPRT expression in ES cells. The resulting HPRT construct should be extremely useful in generating subtle changes in targeted genes.
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