In the early 1980's, technology for transferring DNA sequences of one species into another became available (Costantini e ta l 1981, Harbers ef a/1981, Brinster ef a/1981, 1982). By microinjecting DNA fragments into fertilized oocytes it is possible for heterologous DNA to become integrated into the host genome. The production of transgenic animals has allowed great insight into in vivo regulation of genes and has opened many avenues for research into gene and protein function.
1 .1 1 .2 Expression of Transgene
Microinjected DNA fragments used to make transgenic animals in order to investigate gene expression, usually contain a heterologous reporter gene, the expression of which can be specifically detected, fused either to flanking control sequences specific to the gene being investigated or a gene which is expressed in the cell-type being targetted.
1 .1 1 .3 Reporter genes
Reporter gene sequences used to generate transgenic animals contain the initiation and termination codon as well as sequences essential for translation (Kozak 1985). Reporter genes which have been widely used in transgenic constructs include genes from heterologous species such as the introduction of human, bovine and rat growth hormone genes in mice (Palmiter e ta l 1982,, McGrane e ta l 1988, Russo et al 1988, Hollingshead
et al 1989) which can be recognized by specific antibodies and nucleic acid probes. Other genes of bacterial and viral origin such as lac Z
(R o s s a n t e t a l 1991, Smeyne et al 1992), chloramphenicol acetyltransferase (CAT) (Walker et al 1983, Aronow efa/1995) and the herpes simplex virus thymidine kinase gene (Ross et al 1985), have been used as reporter genes in studies into the expression patterns generated by specific promoter sequences. Many transgenic animals have also been made which express functional transgenes such as specific agonists (Morello e ta l 1986), antagonists (Chen e ta l 1990), antisense (Pepin et al
1992) and oncogenes (Giraldi ef a / 1994), allowing analysis of the systems being investigated and their associated pathologies.
1 .1 1 .4 Proximal promoter sequence.
Whether the constructs used to make transgenic animals are expressed and in which cell-type this expression occurs is dependent on several factors. The proximal promoters of genes transcribed by RNA polymerase II contain a consensus sequence (termed the TATA box) around 30 base pairs upstream of the site of transcriptional initiation (Kornberg 1996). This sequence is required for the binding and activation of the basic transcriptional machinery. It is the site of binding of the TATA box binding protein (TBP) (Nikolov ef a / 1996) which subsequently binds to a number of other proteins known as transcription activation factors (TAFs) prior to the ordered recruitment of general transcription factors and RNA polymerase II to form a preinitiation complex required for transcriptional activation (Roeder ef al 1996). This sequence of events however, only confers basal low level transcription.
1 .1 1 .5 Enhancers and silencers.
Enhancer and silencer elements are gene-specific sequences present in flanking DNA which bind to sequence-specific fra/is-acting transcription factors. The activation or repression of transcription and the level at which this occurs within a certain cell-type is dependent on which transcription factors are present within that cell. The binding of frans-acting factors to enhancer or silencer elements is proposed, via mediator proteins (Bjorklund et al 1996), to interact with the TAFs to promote or repress transcription (Verrijzer ef a / 1996).
These sequences have been found not only in the 5' flanking region of genes but S' to coding sequence (Banerji e ta l 1983, Queen et al
1983) and in intronic sequences (Gillies e ta l 1983). The inclusion of these elements in a transgene construct is essential to the cell-specific expression of the reporter gene (Walker et al 1983, Ornitz et al 1985,
Selden ef a / 1986, Patil efa/1990).
Transgene expression is often obtained in ectopic sites where the gene being targetted is not usually expressed. This is possibly due to the absence of silencer elements in the flanking regions of the trangene which would normally suppress expression of the endogenous gene in that cell- type (Murphy et al 1988) or the presence of a tissue-specific enhancer in the endogenous chromosomal DNA at the site of integration (Banerjee et al 1994). The converse is true of the lack of the expression of a transgene as this could be due to the absence of specific enhancer sequences in the microinjected DNA or the presence of a silencer element at the site of transgene integration. Inappropriate expression of a transgene can also be as a result of the interaction of combinations of regulatory elements from the reporter gene used (within the introns) interacting with those in the flanking sequences. This is exemplified by the unpredictable expression patterns of different foreign genes fused to metallothionein promoter sequences which is dependent on the reporter gene being used (Swanson
et a / 1985).
1 .1 1 .6 Position effect
The effect of the chromosomal DNA at the site of integration on the expression of a transgene is known as position effect and has been widely encountered in transgenic studies. This effect is seen when there are differences in the patterns and levels of transgene expression in a number of transgenic animal lines generated from the same construct (Lacy et al 1983, Al-Shawi et a /1990, Huber et al 1994). Assuming the site of integration of a transgene into a hosts chromosome is random, the differing expression patterns in these animals is due to the interaction between regulatory elements at the integration site and those of the transgene. Each integration event potentially positions a different chromosomal regulatory element juxtaposed to the transgene thereby exerting different effects on its expression.
1 .1 1 .7 Locus control regions.
Regulatory sites within the loci of several genes have been delineated which, when included in a construct used to make transgenic mice impart position-independent, copy-number dependent expression of transgenes (Grosveld et al 1987, Bonifer at al 1990). These sites have been termed the locus control regions (LCR) and are classified by their cell-specific hypersensitivity to DNase I.
The LCR of a gene can comprise of several DNase I hypersensitive regions positioned some distance from the transcriptional start site of the gene (Stief at al 1989, Higgs at al 1990, Jones a ta l 1995). The individual sites may have different functional capacities when used independently (Fraser at al 1990). Some of the regions specifically serve as insulators, confering position-independent expression of the foreign DNA without enhancing or repressing expression (Chung at al 1993, Aronow at al 1995). It appears that these are regions are essential for the establishment of active chromatin domains which are accessible for the proteins necessary for transcription (Steif at al 1989, Forrester at al 1990, B o nife r at al 1991). It is only in combination with other DNase I hypersensitive regions containing the sites for interactions with other trans-
activating factors that the appropriate level of cell-specific expression of a transgene is achieved (Huber ef a/1994).
1 .1 1 .8 Sequences at the 3' end of a transcript
There are sequences at the end of a transcript which are essential for transcriptional termination and processing of the 3' end of the mRNA (Wahle ef a / 1996). These include a polyadenylation signal which causes the polymerization of AMP onto the 3' end of a transcript resulting in a varying length poly A tail. This has been implicated in the control of the stability (Decker at al 1994) and translation of the mRNA (Sachs ef al
1993). In a transgene construct these sequences can either be present as part of the sequence of the reporter gene or, in the case of cDNA transgenes, as additional added sequences (Bijvoet ef a / 1996).
It seems that for cell-specific high level expression of a transgene, the construct must include regions which insulate the sequence from
g,
position effects, cell-specific enhancer^silencer elements and sequences responsible for mRNA processing and correct transcriptional initiation and termination.
1 .1 2 Uses of transgenic animals.
The uses of transgenic animals over the last decade have been wide ranging, including many fields of biological research, for research into topics as varied as the control of gene transcription to the generation of strains of commercially viable agricultural animals (Ward ef a / 1993).
Transgenic technology has been especially important for the study of gene regulation for which adequate cell lines are not available. These studies have a great advantage over those using cell cultures as the expression and physiological regulation of a transgene reflects the regulatory sequences actually utilized in vivo in specific cell-types rather than those sequences which potentially can be used in vitro.
The sequences comprising the LCR and enhancer and silencer elements of many genes have not yet been mapped. Many experiments, therefore, have resulted in inappropriate expression of transgenes. These results, however, have enabled a comparison of promoter sequences used with the results generated leading to the delineation of individual c/s- acting regulatory elements flanking a gene. A complex picture of interacting sequences can therefore be built up enabling a greater understanding the c/s-acting elements which are involved in the regulation of gene expression.
The use of functional transgenes has allowed the introduction of agonist, antagonists, antisense and other molecules to cause specific perturbations in endogenous systems allowing explorations into function and regulation. This has and will in the future, allow specific, non-invasive, changes for which no physiological technique or pharmacological agent provide the same degree of specificity or accuracy. The animals produced
under such rationales, however, are not without their unpredictable drawbacks. Such is the case for early investigations into the growth hormone system. Over-expression of growth hormone under the control of a ubiquitous promoter allowed the production of transgenic mice with significant increases in growth (Palmiter et al 1982). It was hoped that these results could be repeated in farm animals leading to an increase in their meat yields. This was not the case however, the expression of heterologous growth hormone in sheep and pigs did result in increased growth but had severe detrimental physiological effects (Pursel ef a/1989).
The advent of greater understanding into the regulation of transgenes has allowed more accurate targetting of reporter genes with specific flanking sequences. The growth hormone (GH) axis is just one such example of a system extensively investigated using transgenic technology. Specifically targetting cell-types and proteins involved in the regulation of this hormone has permitted, amongst other things, insights into GH protein structure and function (Chen et al 1990), the intracellular mechanisms occurring in somatotrophs (Burton et al 1991, Struthers et al
1991) and the roles of growth hormone-releasing hormone (GHRH) (Mayo ef a / 1988) and insulin-like growth factor I (IGF-I) (Behringer ef a / 1990, Asa ef a / 1992) in the modulation of GH function.
Trangenic animal technology has also been widely used in order to produce disease models for research into specific pathologies. Transgenic mice have been generated expressing toxic and disruptive proteins within targetted cell-types (Behringer e ta l 1987, Borelli e ta l
1989). This has resulted in the production of animals with significantly decreased amounts of proteins such as GH where the cell-type synthesizing these peptides has been disrupted. The phenotypes of these mice have been explored during research into diseases which exhibit the same deficiencies. Similarly, the overproduction of proteins by specifically targetted transgenes has led to animal models for pathologies involving the excessive production of proteins by either tissue hyperplasia or inappropriately by tumors (Quaife et al 1989). For example, pituitary
adenomas caused by the overproduction of growth hormone releasing factor for which a transgenic mouse model has been generated (Asa e ta l
1992).
Transgenesis has been extensively used in the study of cancer (Coletta e ta l 1995). By specifically targetting the expression of transgenes involved in the establishment of tumorigenesis, it has been possible to transform certain cell-types into cancerous states allowing the regulation of these oncogenes and their protein products to be studied (Rindi et al
1989, Kim et al 1991, Eades-Perner et al 1994, Chooi e ta l 1996). Another consequence of specifically targetting oncogene expression in transgenic animals has been the immortalization of cell-lines for use in in vitro studies. (Mellon efa/1990, Jat ef a/1991).
1 .1 3 Choice of species of transgenic animal.
Transgenic technology has extended to several species of domestic and laboratory animals (Mullins ef al 1996). By far the most commonly used is mice due to the well developed methods for the application of this technique and the low cost of maintaining this species.
In recent years several laboratories have been generating transgenic rats (Mullins et al 1990, Zeng e ta l 1994). The efficiency of production of these animals and the protocols used vary between laboratories (Chareau ef al 1996) but there are important advantages for using this species. As well as being relatively cheap to breed and house, the physiology of the rat is much more defined than that of the mouse. This is largely due to their size being compatible with physiological techniques. Surgery used for in vivo bioassays (Bisset et al 1984), multiple-blood sampling procedures (Clark et al 1986) and fine tissue dissections can more easily performed on rats than mice to yield sufficient quantities of material for analysis.
1 .1 4 Transgenic studies of the AVP/OT locus.
generated by the actions of regulatory elements in the flanking sequences of both genes. Expression or its absence is dependent on the interaction of these elements with cell-specific frans-acting factors. In order to delineate these sequences, in the absence of adequate, neuronally derived cell lines which express AVP or OT, transgenesis has been used (Grant et al 1993, Gainer et al 1995, Murphy et al 1995). Expression patterns generated by transgene constructs based on the AVP/OT locus are depicted in figures
1.2 and 1.3.
1.14.1 Bovine AVP (bAVP) expression in transgenic mice.
Initial experiments using 1.25kb of 5' promoter sequence of the bovine AVP gene (bAVP) and the simian virus 40 (SV40) large T-antigen reporter gene targetted expression to the anterior pituitary and pancreas in trangenic mice (Murphy et al 1987). Tumors developed in three lines of mice carrying this gene as a result of this expression. These tumors showed some of the phenotypic properties of multiple endocrine neoplasia and represented a possible disease model for monitoring neoplastic transformation caused by the expression of the SV40 reporter gene (Hindi
et al 1988). In addition, those of the anterior pituitary provide a model for non-hyperplastic somatotroph tumors (Stefaneau et ai 1992). Although previous experiments with short promoter regions of the elastase and insulin genes (Ornitz et al 1985, Hanahan et al 1985) had conferred cell- specific expression of reporter genes it is obvious that additional sequences are required for appropriate cell-specific bAVP expression.
There are two possible explanations for the expression pattern generated with this construct. It is possible that the 1.25kb of bAVP promoter does not include enhancer elements to activate expression in vasopressinergic cells and silencer elements to repress expression in the anterior pituitary and pancreas. It is also possible that the combination of regulatory elements included within the promoter region and reporter gene of this construct is responsible for this expression pattern. Replacing the SV40 reporter gene with the CAT reporter gene causes ubiquitous
expression in nearly all the peripheral tissue examined in two lines of the transgenic mice generated (Ang et al 1993). This indicates that the expression directed by this promoter region is dependent, to some extent, on the reporter gene used.
The addition of the structural gene for bAVP with 200 bp of sequence 3' to exon III to the 1.25 kb promoter fragment conferred a more restricted pattern of expression within the neuronal tissue of 3 lines of mice (Ang eta! 1993). All lines showed hypothalamic expression with additional expression detected in the adrenal medulla in two of the three lines. The addition of the AVP gene and 200bp of 3' sequence, therefore, restricts expression to tissue that is derived from neuronal progenitors. More accurately targetted expression occurred in the PVN and SON but not the parvocellular SON with a larger construct containing 9kb upstream of 5’ flanking sequence, the structural gene and 1.5 kb of 3' sequence. However 3 lines of transgenic mice carrying this transgene also exhibited peripheral expression in the ovary (Ang ef a /1993).
It has been concluded, therefore, that the cell-specific expression of bAVP in transgenic mice is due to repression of expression in non- vasopressinergic cells. It is possible that silencer elements are present within the gene and 200bp of 3' flanking sequence which repress expression in non-neuronal tissue. Additional regulatory elements in the flanking sequeces extending 9kb 5’ of and 1.5 kb 3' of the bAVP gene repress expression in all tissues except the magnocellular vasopressin neurones of the PVN and SON. This piece of DNA however does not contain sufficient regulatory elements to direct expression to parvocellular neurones of the SON. The ovarian and adrenal expression can be explained by the presence of one or more tissue-specific enhancer elements in the foreign DNA present which are usually suppressed by additional elements outside the span of these fragments.
When mice generated with both the smaller (1.25kb 5' to 0.2 kb 3') and larger (9kb 5’ to 1.5 kb 3’) bAVP constructs were salt loaded, an increase in expression but not length of the transgene mRNA in the PVN
and SON was detected (Ang et al 1993). The regulatory element responsible for increased expression due to physiological stimulation (possibly the cAMP responsive element described previously) is therefore present in both constructs.
1 .1 4 .2 Bovine OT (bOT) expression in transgenic mice.
Similar experiments have been performed to delineate the cis- acting elements responsible for the regulated expression of the bOT gene (figure 1.2). A construct consisting of sequences 0.6 kb 5', 1.8 kb 3' and the entire structural gene of bOT reproducibly directed expression to the oxytocinergic cells of the SON and the PVN, the lung and Sertoli cells of the testis in transgenic mice (Ho eta! 1995). The hypothalamic expression was also physiologically regulated with an increase in the abundance of the transgene transcript occurring during dehydration. The Sertoli cells are a site of peripheral expression of the endogenous OT gene in cattle but not in mice. In these mice the level of testicular expression in the transgene is ten-fold larger than that of the endogenous OT in cattle and the resulting transcripts are translated and faithfully processed (Ang e ta ! 1994). This suggests that this construct contains regulatory elements capable of interacting with trans-acWng factors in the mouse testis to recapitulate the bovine expression pattern of OT (Ang ef a/1991). Sequences regulating the levels of testicular expression, however, are absent. No OT expression is detectable in the lungs of cattle indicating the absence of silencer elements repressing the expression of the transgene in this tissue, or the presence of elements which interact with frans-acting factors specific to the mouse lung.
Unlike the bAVP gene, transgenic mice with additional bOT flanking sequences did not result in increased cell-specificity in the expression of the transgene. Three lines of transgenic mice generated with a bOT transgene containing the same amount of upstream promoter sequence (0.6 kb) but with 2.5 kb of downstream sequence did not exhibit hypothalamic expression although transgene transcripts were present in