| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOPICAL REVIEW |
1 Gene Targeting Laboratory, The John Curtin School of Medical Research, Building 131, Garran Road, The Australian National University, Canberra, ACT 0200, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionReferences |
|---|
(Received 18 April 2007;
accepted after revision 10 May 2007;
first published online 10 May 2007)
Corresponding author K. I. Matthaei: Gene Targeting Laboratory, The John Curtin School of Medical Research, GPO Box 334, Canberra City, ACT 0200, Australia. Email: klaus.matthaei{at}anu.edu.au
|
Introduction |
|---|
|
TopAbstractIntroductionReferences |
|---|
Problems with transgenic mice due to random integration
The generation of the first transgenic mouse by injection of a DNA construct directly into the pronucleus of a fertilized single cell mouse embryo (Palmiter et al. 1982) has resulted in the generation of thousands of such strains. Importantly, the site at which the DNA is integrated is random, as are the number of copies of the transgene. Although the expression of the construct is faithful for the promoter, on many occasions it may also be significantly influenced by the local environment at the integration site (the ‘position’ effect). This can lead to the promiscuous expression of the transgene (often referred to as ‘leakiness’), due to modification of the specificity of the promoter, or at times to a more severe phenotype, due to disruption of an unknown gene by insertion of the transgene (insertional mutagenesis). A number of different ‘founder’ animals with different copy numbers and different integration sites must therefore be assessed in order to determine the correct/faithful expression of each transgene. Surprisingly, in one example, 24 different founders resulted in 24 different expression patterns (Feng et al. 2000) making it impossible to determine which pattern was correct.
One way to overcome this problem is to use a ‘knock-in’ procedure where the transgene is introduced into a specific locus using homologous recombination in embryonic stem cells. In this situation a modified gene can be expressed under its own promoter (see for example Yang et al. 2006), or a transgene can be expressed from a ubiquitous promoter, such as Rosa26 (Soriano, 1999). An alternative approach to improve promoter specificity is to use larger DNA fragments, which can be manipulated in bacterial (BAC) or yeast (YAC) artificial chromosomes, to drive gene expression. These larger fragments can avoid the position effect since they contain more of the local environment of the promoter thereby ensuring its ability to function normally. These fragments can vary in length up to 250 kb as opposed to the 1–2 kb commonly used for transgenic constructs and they result in faithful control of reporter gene expression (see for example Moreira et al. 2006). Indeed a major initiative using this approach estimates that 85% of BAC transgenic constructs express reproducibly in multiple transgenic lines (Gong et al. 2003), thereby reducing the need to generate and assess more than a few lines to identify faithful expression. However, it is not clear whether the presence of other genes, transcription factors, microRNAs or control regions in these fragments may compromise results. In the BAC studies mentioned above, the readout was expression of a reporter gene (EGFP) and not the expression of a functional protein such as a particular cytokine that can produce specific effects. It would be interesting to compare the phenotype of the interleukin(IL)5 transgenic mouse that uses a minimal promoter (Dent et al. 1990) with a mouse expressing IL5 from a BAC which would also contain a number of other cytokines due to their very close proximity, including IL3, IL4, IL13 and granulocyte macrophage colony stimulating factor (Lee & Young, 1989; McKenzie et al. 1999; Avery et al. 2004). This approach, without careful analysis, may therefore not be as widely applicable as first thought and the ‘knock-in’ procedure, although more complex, may still be the method of choice. Again, caution must be taken since the ‘knock-in’ will produce an insertional deletion of the gene controlled by the promoter to be used (see for example Gazit et al. 2006). Moreover, even in the heterozygous state the mice can have a phenotype if the gene exhibits haploinsufficiency (deletion of one functional allele resulting in modified gene expression). However, the insertional deletion can be avoided if the construct uses an internal ribosome entry site (IRES) followed by the transgene and these are placed between the stop codon and the polyadenylation signal of the target gene. In this case a bicistronic transcript is made containing both genes that are then translated into two separate proteins (see for example Rees et al. 1996), resulting in the expression of the transgene without disrupting the endogenous gene.
However, the potential ‘leakiness’ of conventional transgenic mice complicates most current studies involving genetic manipulations, as we will see in the following sections.
Tissue and temporal specific control of gene function
CreLoxP. The deletion of a gene results in embryonic lethality if there is an absolute requirement for that gene during development. This lethality can be avoided if the gene is deleted later in life or if it is deleted in a tissue-specific manner particularly if the gene has multiple functions. The most common method to obtain tissue-specific control of gene deletion is the CreLoxP system modified from bacteriophage. LoxP sites are short DNA sequences that are recognized by a specific DNA recombinase enzyme called Cre (causes recombination) that deletes any DNA between the two sequences (see Fig. 1). An exon in the gene of interest is flanked by these LoxP sites and the modified gene is introduced into its correct location in embryonic stem cells by homologous recombination. The stem cells are injected into mouse blastocysts and mice generated (for a fuller description of chimæra generation see Matthaei, 2004) in which the gene is ‘floxed’ but fully functional until it is inactivated by the Cre recombinase due to the removal of the exon. This is achieved by breeding the ‘floxed’ mice with a mouse transgenic for the Cre recombinase usually under the control of a tissue-specific promoter (first described in Gu et al. 1994, but see also Rajewsky et al. 1996; Kwan, 2002). The result is deletion of the gene in a specific tissue.
|
Notwithstanding the various drawbacks inherent in the use of conventional transgenic mice, the major failing with the Cre recombinase system is that it is not reversible.
Reversible tissue and temporal specific control of gene over-expression
Tetracycline response system. Reversible control of gene expression has for some time been possible with the tetracycline response system (Kistner et al. 1996). In this procedure, one mouse strain is made to express a tetracycline-responsive transactivator protein, tTa (tet-off) or rtTa (tet-on), usually under the control of a tissue-specific promoter. A second mouse expresses the gene of interest under the control of a minimal promoter that requires the transactivator protein (tTA or rtTA) and tetracycline. Doubly transgenic mice for both transgenes can then be regulated by the addition (tet-on) or removal (tet-off) of tetracycline in the drinking water allowing control of the gene of interest in a tissue- and temporal-specific manner.
However, this system again is based on conventional transgenic mice and for the reasons given above can also suffer from promiscuous expression. A particular problem occurs due to the lack of complete silence of the minimal promoter when the transactivator protein is absent, hence the system is never fully ‘off’. More recently, a major improvement for the rtTA system was developed involving a second protein (tTS) that is a fusion of the transactivator protein and the KRAB-AB silencing domain of the Kid-1 protein (Zhu et al. 2002). tTS binds to the minimal promoter in the absence of tetracycline and ensures complete silencing of the promoter (Zhu et al. 2002). In the presence of tetracycline, tTS dissociates from the promoter allowing rtTa to bind and drive transcription allowing better temporal control. However, it is important to note that tetracycline is an antibiotic that could affect the bacterial flora of the experimental mice necessitating multiple control groups. Moreover the recent finding that the rtTA protein itself can produce emphysema-like symptoms in the lungs of mice is a further complication (Sisson et al. 2006). Whether the tTS protein also causes similar symptoms in the lung or indeed if there is a general problem of expressing rtTA or tTS protein in different tissues needs clarification.
In spite of these potential problems, the tetracycline system has enabled tissue and temporal control of gene function.
LacO/LacIR system. Recently, an alternative, reversible and inducible expression system based on the lac operon of E. coli was developed for use in mammals (Cronin et al. 2001). In E. coli, the lac operon functions by a repression mechanism involving the production of an inhibitor protein LacIR, that binds to regulatory sites lacO in the promoter and turns off transcription of the genes required for lactose metabolism. By adding lactose, the LacIR protein changes its binding affinity for the lacO sequences, dissociates and transcription of the lac genes can occur. E. coli uses this system to tightly control the genes required for the utilization of lactose and it is completely reversible. In a proof of principle, Scrable and colleagues were able to modify the tyrosinase promoter with LacO sites and successfully switch coat colour on and off at will when IPTG (an analogue of lactose) was added to the drinking water of mice that also expressed the LacIR protein (Cronin et al. 2001). Tyrosinase activity could also be regulated during discrete periods of embryogenesis (Cronin et al. 2003).
In a subsequent study, similar control of gene expression during embryogenesis was demonstrated in vivo for a luciferase reporter gene under the control of the ubiquitous Huntingtin promoter with introduced LacO sites (HuntingtinLacO-luciferase) (Ryan & Scrable, 2004). In this study it was shown that luciferase activity could be regulated in live offspring in utero by the addition of IPTG to the drinking water of the pregnant dam (Ryan & Scrable, 2004).
In order to study the regulation of this gene in different tissues we obtained mice expressing the HuntingtinLacO-luciferase (HDOSluc) reporter and mice expressing the LacIR repressor protein from Professor Scrable. By crossing these mice we have observed repression of luciferase expression by the LacIR protein to less than 10% in a range of different tissues (Fig. 2A and Matthaei et al. 2006). Importantly, addition of IPTG to the drinking water produced de-repression (re-activation) of luciferase expression within 48 h to 80% of normal activity (Fig. 2B for kidney). This level of repression is similar to that achieved with siRNA (Carmell et al. 2003) but without side-effects in non-target tissues (Jackson et al. 2003). We therefore see tight reversible regulation of a transgene using a common compound (sugar) that should have minimal side-effects. This system warrants intensive investigation since it possibly provides the best method of controlling gene overexpression or deletion (see below).
|
Epigenetics is the transmission of information from a cell or multicellular organism to its descendants without that information being encoded in the nucleotide sequence. Evidence is now emerging that epigenetic changes can occur due to the transmission of RNA or protein in the oocyte or sperm from transgenic parents to non-transgenic offspring. We were first alerted to this problem when we were crossing a ‘
-galactosidase Stop floxed EGFP’ mouse (see Fig. 1) with Cre mice controlled by the Tnap promoter (Lomeli et al. 2000). In this cross we expected to see the Cre delete the -galStop and allow expression of EGFP (Figs 1 and 3A). Indeed, when the Cre transgenic parent was the male we did see faithful transmission of the Cre transgene to the offspring and deletion of our floxed gene in these mice (Cre/delLoxP, Fig. 3A). However, when the Cre-carrying parent was the female, we saw deletion of our floxed gene in all offspring, even in those that had not inherited Cre (WT/delLoxP, Fig. 3B). This phenomenon has also been observed by others. Tissue-specific deletion occurred when the Cre transgene controlled by the keratin K5 promoter was carried by the male parent, but deletion in all offspring when the Cre transgene was carried by the female parent (Ramirez et al. 2004), including those not inheriting Cre. Moreover, when the Cre parent was female the deletion was no longer tissue specific but occurred in all tissues of the offspring, suggesting deletion very early during embryogenesis (Ramirez et al. 2004). These data suggest that RNA or Cre protein may be present in all oocytes of a Cre transgenic female and this is transmitted to non-transgenic offspring causing generalized deletion. Moreover, since this phenomenon has been seen for two different promoters driving Cre, suggests that the effect may be more general. In this case, non-transgenic littermates would not constitute an appropriate control.
|
|
The way forward
Future gene-deficient mice. The ideal system to test the effects of a gene deletion should incorporate complete reversibility and no non-specific side-effects. Currently none of the commonly used methods satisfy these requirements since they rely on the use of transgenic mice with their inherent limitations. Our solution to this dilemma is to insert LacO sites into the promoter of our gene of interest by homologous recombination like those employed to introduce LoxP sites. By crossing these LacO mice with the ubiquitous LacIR-expressing mice, generalized gene expression can be regulated by the addition and removal of IPTG in the drinking water. Moreover, this system will also allow the regulation of mutated forms of genes of interest. In this case the mutation as well as the LacO sites will be introduced into the germline by homologous recombination, then regulated by LacIR and IPTG as above. Tissue-specific regulation of the gene can be obtained by expression of the LacIR protein driven from tissue-specific promoters. However, to avoid the problem of position effect, ‘knock-in’ methods of LacIR into tissue-specific promoters using homologous recombination should be implemented using IRES constructs as indicated above.
Future transgenic mice. The ideal expression of a transgene should be totally tissue specific, not modify the host genome or be modified by the host genome, and be completely reversible. All current methods apart from site-specific insertions (see above) potentially suffer from modification by the local environment. Moreover, the only commonly used reversible system using tetracycline also suffers from side-effects from the tTA protein and possibly from the use of the antibiotic (see above). To overcome these problems, we are currently trialing a new method in which we are integrating our transgenes of interest into episomes (D. Rangasamy & K. I. Matthaei, unpublished observations). Episomes are circular, self-replicating DNAs often of viral origin that are stably and faithfully inherited during mitosis but are not integrated into the chromosomes of mammalian cells. Moreover, since they are not integrated, episomes carrying a transgene should not only avoid the ‘position’ effect from neighbouring genes but also not cause insertional mutation of an unknown gene thereby making them a possibly ideal transgene carrier.
Naturally occurring episomes are large since they require the production of an array of viral proteins to allow faithful replication and segregation into daughter cells (herpesvirus for example is 172 kb; Wade-Martins et al. 2000). This makes the genetic manipulation of episomes difficult. However, recently it was found that only one of the multiple proteins is necessary for episomal replication, the scaffold/matrix region of the human -interferon gene cluster (Piechaczek et al. 1999) resulting in a plasmid of only 6.7 kb. Moreover this episome avoids insertional mutagenesis and methylation (Jenke et al. 2004) suggesting that it is an ideal transgene carrier. It is our intention to use the LacO/LacIR control and episomal expression of the transgene to generate transgenic mice that have fully reversible, tissue-specific and temporal control of transgene expression without modification from the host cell. If successful, this will be a major breakthrough for the generation of transgenic mice.
Conclusion
It is not my intention to suggest that all genetically modified mouse experiments are flawed or that useful information has not been generated from genetically altered mice. I have also limited my discussion to situations where tissue and temporal control of gene expression is required. Simple deletions of gene function will continue to have a place in some situations as will transgenic mice using minimal promoters. However, it is imperative to understand the limitations of these systems so that appropriate controls can be included and correct conclusions can be made. It is for this reason that Hagg (1999) should be congratulated. He was one of the first to point out that there was a problem with mixed genetic backgrounds in gene deletion experiments. Hagg and colleagues had published a specific phenotype of the deletion of the p75 nerve growth factor receptor (Van der Zee et al. 1996) but later discovered that the background strains were responsible for the phenotype and not the gene deletion. The senior author therefore corrected the literature by retracting the original result (Hagg, 1999). Many other published data may be similarly affected by previously unforseen problems. My intention here is to alert the reader to such possibilities in order to make the best use of a powerful tool which can be used to better understand gene expression and function and thereby health and disease. At the same time, discussions like these might stimulate the reader to re-assess their data in the light of some of the complications presented here, particularly when ‘inexplicable’ results are obtained.
In conclusion, the potential to use completely reversible methods like the LacO/LacIR system will allow each experimental group of animals to be their own control and should help detect epigenetic (or other) influences in studies of both gene overexpression and gene deletion thereby providing exciting new developments for the future.
|
References |
|---|
|
TopAbstractIntroductionReferences |
|---|
Blackburn AC, Matthaei KI, Lim C, Taylor MC, Cappello JY, Hayes JD, Anders MW & Board PG (2006). Deficiency of glutathione transferase zeta causes oxidative stress and activation of antioxidant response pathways. Mol Pharmacol 69, 650–657.
Carmell MA, Zhang L, Conklin DS, Hannon GJ & Rosenquist TA (2003). Germline transmission of RNAi in mice. Nat Struct Biol 10, 91–92.[CrossRef][Medline]
Cronin CA, Gluba W & Scrable H (2001). The lac operator-repressor system is functional in the mouse. Genes Dev 15, 1506–1517.
Cronin CA, Ryan AB, Talley EM & Scrable H (2003). Tyrosinase expression during neuroblast divisions affects later pathfinding by retinal ganglion cells. J Neurosci 23, 11692–11697.
Dent LA, Strath M, Mellor AL & Sanderson CJ (1990). Eosinophilia in transgenic mice expressing interleukin 5. J Exp Med 172, 1425–1431.
Faast R, Thonglairoam V, Schulz TC, Beall J, Wells JR, Taylor H, Matthaei K, Rathjen PD, Tremethick DJ & Lyons I (2001). Histone variant H2A.Z is required for early mammalian development. Curr Biol 11, 1183–1187.[CrossRef][Medline]
Feng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, Wallace M, Nerbonne JM, Lichtman JW & Sanes JR (2000). Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51.[CrossRef][Medline]
Gazit R, Gruda R, Elboim M, Arnon TI, Katz G, Achdout H, Hanna J, Qimron U, Landau G, Greenbaum E, Zakay-Rones Z, Porgador A & Mandelboim O (2006). Lethal influenza infection in the absence of the natural killer cell receptor gene Ncr1. Nat Immunol 7, 517–523.[CrossRef][Medline]
Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME & Heintz N (2003). A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425, 917–925.[CrossRef][Medline]
Gu H, Marth JD, Orban PC, Mossmann H & Rajewsky K (1994). Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265, 103–106.
Hagg T (1999). Neuronal cell death: retraction. Science 285, 340.[Medline]
Hendry IA, Hill CE & McLennan IS (1987). RU38486 blocks the steroid regulation of transmitter choice in cultured rat sympathetic ganglia. Brain Res 402, 264–268.[CrossRef][Medline]
Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B, Cavet G & Linsley PS (2003). Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21, 635–637.[CrossRef][Medline]
Jenke AC, Scinteie MF, Stehle IM & Lipps HJ (2004). Expression of a transgene encoded on a non-viral episomal vector is not subject to epigenetic silencing by cytosine methylation. Mol Biol Rep 31, 85–90.[CrossRef][Medline]
Kellendonk C, Tronche F, Monaghan AP, Angrand PO, Stewart F & Schutz G (1996). Regulation of Cre recombinase activity by the synthetic steroid RU 486. Nucl Acids Res 24, 1404–1411.
Kistner A, Gossen M, Zimmermann F, Jerecic J, Ullmer C, Lubbert H & Bujard H (1996). Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Natl Acad Sci U S A 93, 10933–10938.
Kwan KM (2002). Conditional alleles in mice: practical considerations for tissue-specific knockouts. Genesis 32, 49–62.[CrossRef][Medline]
Lee JS & Young IG (1989). Fine-structure mapping of the murine IL-3 and GM-CSF genes by pulsed-field gel electrophoresis and molecular cloning. Genomics 5, 359–362.[CrossRef][Medline]
Lim CEL, Matthaei KI, Blackburn AC, Davis RP, Dahlstrom JE, Koina ME, Anders MW & Board PG (2004). Mice deficient in glutathione transferase zeta/maleylacetoacetate isomerase exhibit a range of pathological changes and elevated expression of alpha, mu, and pi class glutathione transferases. Am J Pathol 165, 679–693.
Lomeli H, Ramos-Mejia V, Gertsenstein M, Lobe CG & Nagy A (2000). Targeted insertion of Cre recombinase into the TNAP gene: excision in primordial germ cells. Genesis 26, 116–117.[CrossRef][Medline]
McKenzie GJ, Fallon PG, Emson CL, Grencis RK & McKenzie AN (1999). Simultaneous disruption of interleukin (IL)-4 and IL-13 defines individual roles in T helper cell type 2-mediated responses. J Exp Med 189, 1565–1572.
Matthaei KI (2004). Caveats of gene targeted and transgenic mice. In Handbook of Stem Cells, ed. Lanza R, Gearhart J, Hogan B, Melton DW, Pederson R, Thomson J & West W, pp. 589–598. Elsevier Inc., San Diego, CA, USA.
Matthaei KI, Lam KE, Grayson TH, Scrable H & Hill CE (2006). Tissue and temporal control of gene function in vivo. Transgenic Res 15, 777.
Moreira PN, Pozueta J, Giraldo P, Gutierrez-Adan A & Montoliu L (2006). Generation of yeast artificial chromosome transgenic mice by intracytoplasmic sperm injection. Methods Mol Biol 349, 151–161.[Medline]
Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC & Evans RM (1982). Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300, 611–615.[CrossRef][Medline]
Piechaczek C, Fetzer C, Baiker A, Bode J & Lipps HJ (1999). A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells. Nucl Acids Res 27, 426–428.
Rajewsky K, Gu H, Kuhn R, Betz UA, Muller W, Roes J & Schwenk F (1996). Conditional gene targeting. J Clin Invest 98, 600–603.[Medline]
Ramirez A, Page A, Gandarillas A, Zanet J, Pibre S, Vidal M, Tusell L, Genesca A, Whitaker DA, Melton DW & Jorcano JL (2004). A keratin K5Cre transgenic line appropriate for tissue-specific or generalized Cre-mediated recombination. Genesis 39, 52–57.[CrossRef][Medline]
Rassoulzadegan M, Grandjean V, Gounon P, Vincent S, Gillot I & Cuzin F (2006). RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse. Nature 441, 469–474.[CrossRef][Medline]
Rees S, Coote J, Stables J, Goodson S, Harris S & Lee MG (1996). Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein. Biotechniques 20, 102–104, 106, 108–110.
Ryan A & Scrable H (2004). Visualization of the dynamics of gene expression in the living mouse. Mol Imaging 3, 33–42.[CrossRef][Medline]
Sisson TH, Hansen JM, Shah M, Du Hanson KEM, Ling T, Simon RH & Christensen PJ (2006). Expression of the reverse tetracycline-transactivator gene causes emphysema-like changes in mice. Am J Respir Cell Mol Biol 34, 552–560.
Soriano P (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21, 70–71.[CrossRef][Medline]
Thyagarajan B, Guimaraes MJ, Groth AC & Calos MP (2000). Mammalian genomes contain active recombinase recognition sites. Gene 244, 47–54.[CrossRef][Medline]
Van der Zee CE, Ross GM, Riopelle RJ & Hagg T (1996). Survival of cholinergic forebrain neurons in developing p75NGFR-deficient mice. Science 274, 1729–1732.
Wade-Martins R, White RE, Kimura H, Cook PR & James MR (2000). Stable correction of a genetic deficiency in human cells by an episome carrying a 115 kb genomic transgene. Nat Biotechnol 18, 1311–1314.[CrossRef][Medline]
Yang T, Riehl J, Esteve E, Matthaei KI, Goth S, Allen PD, Pessah IN & Lopez JR (2006). Pharmacologic and functional characterization of malignant hyperthermia in the R163C RyR1 knock-in mouse. Anesthesiology 105, 1164–1175.[CrossRef][Medline]
Zheng B, Zhang Z, Black CM, de Crombrugghe B & Denton CP (2002). Ligand-dependent genetic recombination in fibroblasts: a potentially powerful technique for investigating gene function in fibrosis. Am J Pathol 160, 1609–1617.
Zhou Z, Wang D, Wang XJ & Roop DR (2002). In utero activation of K5.CrePR1 induces gene deletion. Genesis 32, 191–192.[CrossRef][Medline]
Zhu Z, Zheng T, Lee CG, Homer RJ & Elias JA (2002). Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling. Semin Cell Dev Biol 13, 121–128.[CrossRef][Medline]
|
Acknowledgements |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
