Primary contributions to this chapter were made by J.D. Marth (HHMI, University of California at San Diego).

MANY CELL LINES THAT LACK GLYCOSYLTRANSFERASES or glycosidases do not exhibit obvious phenotypes, whereas identical mutations in intact organisms yield pathologies and in some cases embryonic lethality. Several approaches to determine oligosaccharide function in vivo include expressing glycosidases, masking glycosyltransferases, competing glycosyltransferases, and overexpressing endogenous glycosyltransferases (Figure 33.1). This chapter details approaches that are useful in defining the function of glycans, especially by transgenesis and gene targeting using the mouse as a model vertebrate. The specific techniques used are reviewed. A significant amount of information on glycan function has been gained in recent years using these approaches, and this information is also presented.

Figure 33.1

Approaches to altering glycosylation in studies of oligosaccharide function. (Modified, with permission, from [66] Varki and Marth 1995.)

Background

Following the development of molecular genetic techniques to isolate and alter DNA structure in vitro, methods to alter the germ-line DNA of intact animals were devised. In the early 1980s, a procedure was successfully developed in the mouse, referred to as transgenesis, that introduces exogenous DNA into the germ line. The term transgenesis refers to the production of a transgenic organism, e.g., an organism in which DNA from another organism has been transferred, so that the host acquires the genetic traits encoded by the transferred genes in its chromosomal composition. The mouse was considered the best organism in which to initially develop transgenesis, since all mammals have similar physiologies and mice can be maintained easily in relatively little space. Additionally, mouse generation time is short, approximately 8 weeks, which is rapid enough for practical use. Why transgenic animals? Gene transfer is valuable in diploid organisms with long life cycles where classic methods (breeding) are impractical. In addition, studies of some systems (the immune system, for example), or of ontogeny itself, require a physiologic “body.”

In developing transgenesis, the earliest studies used blastocyst-stage mouse embryos for injection of SV40 into the blastocoel cavity. After implantation in foster mothers, live-born offspring were found to contain the SV40 genome in various tissues. However, the intact SV40 DNA did not integrate or colonize the germ line, and thus, germ-line transformation did not occur. Although retroviral transduction of blastocyst embryos did achieve germ-line modification, expression was poor, due to the now recognized presence of cellular factors that inhibit retroviral expression. Subsequently, the development of pronuclear microinjection techniques using purified DNA resulted in germ-line transformation and transgene expression. Transgene expression can be regulated by the promoter-enhancer elements chosen in constructing the transgene vector. Transgenesis by pronuclear injection results in an animal in which the transgene is most frequently integrated in all cells of the body. This procedure is effective in providing for dominant genetic studies in vivo.

An approach to generating specific recessive genetic lesions in vivo in intact vertebrates has long been sought. Understanding the physiologic function of a gene may be best gained by studying an organism rendered deficient in the function of that particular gene. In the 1980s, a combination of techniques and reagents was developed that allowed the production of recessive genetic lesions in intact mice. One development was the invention of PCR. Separately, the derivation of pluripotent embryonic stem cell lines allowed the production of chimeric mice with modified germ lines. Recessive genetic models are now commonly produced by a method often referred to as gene targeting. A refinement to this approach has also been gained by development of site-specific DNA recombination during and subsequent to gene targeting. Genetic lesions can now be generated in specific cell types of intact mice and at specific times.

Transgenesis ^(1–8)

To produce transgenic animals requires some knowledge of the organism's embryonic development, as well as competence in microinjection techniques and various surgical procedures. In addition, to assess the validity of results achieved by transgenesis requires an understanding of how transgenes affect endogenous genomic structure and how they become expressed. Overall, the technique is rather complex, expensive, and inefficient. The production of a transgene vector that incorporates the desired elements is relatively easy. Transgene DNA is microinjected into a pronucleus of a fertilized egg (zygote), prior to the fusion of male and female pronuclei (Figure 33.2). The next day, zygotes that have survived the injection and have become two-cell-stage embryos are implanted into the oviduct of a female mouse that has been prepared to accept an embryo by prior mating with a vasectomized male. It can require approximately 100 zygotes to generate one transgenic mouse. On average, 50% of zygotes survive DNA injection and divide to the two-cell stage. Of those, 50% may implant successfully and develop to term. Anywhere from 5% to 25% (1–6 mice from 100 zygotes) may be transgenic as usually determined by genotyping tail DNA.

Figure 33.2

Pronuclear injection of DNA into a mouse zygote during the production of transgenic mice.

The mechanism of DNA integration likely involves breaks in chromosomal DNA, since linear DNA is fivefold more efficient at integration than is circular DNA. Integration is rapid, occurring more than 90% of the time while embryos are at the one-cell stage. If integration occurs at the two-cell stage, the transgenic mouse will be chimeric, because not all cells in the body will have acquired the transgene. Transgenes integrate predominantly as head-to-tail arrays of anywhere between one and several hundred copies. Approximately 5% of transgene integrations occur within an endogenous gene and thus generate a break, and potentially a mutation of a functional cellular gene. Insertional mutation events producing an inactivating (null) mutation may lead to observable phenotypes that are not due to transgene expression. This is the reason that multiple transgenic lines established by unique founders (the “founding” transgenic mouse is the original born from a specific pronuclear injection) are considered essential to control for the effect of the transgene on the endogenous genome.

Transgene expression is dependent on a number of factors, and early experiments have given significant insights into chromosomal structure and function. Perhaps only 10–50% of transgenic mice express the transgene, which was originally much lower before it was recognized that the presence of plasmid sequences in transgenes is detrimental. It appears that host factors are able to extinguish expression of transgenes incorporating bacterial and bacteriophage DNA sequences. Most, but not all, transgenes examined are appropriately expressed, considering their use of specific promoter and enhancer elements. Since transgene expression can be higher than that of an endogenous homolog, optimal gene expression does not depend on normal chromosomal context. However, chromatin structure at the site of integration can influence transgene expression. Levels of expression vary with distinct lines of equal copy number and different integration site, inferring that chromosomal position can influence expression. This may be due to the varied accessibility to host transcription factors. In addition, a few transgenic lines may not express at all, perhaps due to transgene integration into regions of heterochromatin. Because multiple transgene copies are present in the arrays, there is no way to determine the number of transcriptionally active transgenes present. However, the poor correlation of transgene expression with transgene copy number implies that only a few of the transgenes are expressed and that the array is sensitive to chromosomal position.

Tissue-specific transgene expression can be routinely achieved. In general, if a transgene is expressed at all, it is usually appropriately expressed, despite its integration at different chromosomal positions. Therefore, trans-acting proteins involved in establishing host-tissue-specific gene expression can find their target sequences and activate transcription at most chromosomal positions. This appears to be true even among species divergent by millions of years of evolution. Therefore, many signals involved in tissue-specific gene regulation are evolutionarily conserved. Occasionally, sequence effects in transgene vectors comprising elements from divergent sources result in expression patterns not observed with either the gene or promoter/enhancer element separately. Promoter and/or enhancer elements normally expressed in many cell types are often the most sensitive, in transgenes, to the influence of chromosomal position on expression. With a tissue-specific enhancer, the chromosomal position can only influence expression levels in one or a few cell types. However, with a promoter/enhancer that functions in a variety of cell types, the chromosomal position effect may vary in different cell types and from one founder animal to another, leading to an apparently random success rate in establishing transgene expression. Some transgenes have never been successfully expressed, which may be due to embryonic lethality with transgene expression. Gene “silencers” have also been reported, including the 3′ region of the v-src-coding sequence and a CD4 gene intron that controls T-cell lineage commitment.

Transgene expression in offspring is generally identical to that of founder parents. However, alterations have been observed as a result of genomic imprinting. Genomic imprinting breaks the Mendelian rule of haploid equivalency and was first recognized in studies of balanced Robertsonian chromosomal translocations, where some alleles must be inherited from both male and female genomes for embryo viability. The mechanism of genomic imprinting includes changes in DNA methylation. Transgenes can also undergo imprinting, leading to variations in expression from one generation of offspring to the next, depending on the sex of the parent providing the transgene.

Modifying Glycosylation in Vivo by Transgenesis ^(9–17)

In the first transgenic experiment devised to alter glycosylation in vivo, an influenza virus sialic acid O-acetylesterase was found to interfere with embryogenesis, as its expression resulted in a developmental block at the two-cell stage (Figure 33.3). This viral enzyme removes the O-acetyl group linked to many sialic acids contained on Golgi and cell surface oligosaccharides by an endogenous sialic acid O-acetyltransferase activity (see Chapter 15). When the transgenic O-acetylesterase was expressed preferentially in somatic compartments, alterations in tissue morphogenesis were observed in the retina and adrenal gland. These results imply that the O-acetylation of sialic acids is an oligosaccharide modification required for early preimplantation embryogenesis and in organogenesis.

Figure 33.3

Developmental block at the two-cell embryo stage induced by the influenza sialic acid O-acetylesterase following pronuclear injection. (Reprinted, with permission, from [16] Varki et al. 1991 [© Cell Press].)

The above example demonstrates the use of an esterase to remove an endogenous oligosaccharide modification (Figure 33.1). This is distinct from an approach to overexpress an endogenous glycosyltransferase in an effort to notably enhance endogenous function. In studies with a β1–4 galactosyltransferase transgene, overexpression led to reduced sperm-egg binding and inhibited the development and lactation response of the mammary gland. This β1–4 galactosyltransferase is expressed in the Golgi of most cells normally, but it can also be found on the sperm cell surface where it has been implicated in regulating sperm-egg binding during fertilization. Ectopic expression of either α1–3 galactosyltransferase (Gal3T) or α1-3/4 fucosyltransferase (FucT-III) had no effect on development, although the expected galactosylated or fucosylated glycoconjugates were found in higher abundance in adult tissues. With overexpression of Gal3T, mice were reported with lower body weights, hair growth alterations, and increased mortality. With increased α1-3/4 fucose linkages engineered in the stomach of a transgenic mouse, the desired increase in Lewis b antigen was observed and may provide a mouse model for studies of infection with Helicobacter pylori, a bacterium known to cause stomach ulcers.

Masking the action of a “competing” glycosyltransferase may be effected by overexpression of a glycosyltransferase or glycosidase. Formation of the α1–3Gal terminus on oligosaccharides by Gal3T generates the major xenotransplantation antigen of human relevance. Old World primates have specifically lost the ability to generate this structure from acquired germ-line mutations in the Gal3T gene. As a result, human serum is rich in immunoglobulins that bind to this epitope and initiate the immunologic rejection of organs transplanted from other species, such as pigs. A means to reduce or eliminate cell surface α1–3Gal residues was desired, and one means to achieve this involved overexpression of an α1–2 fucosyltransferase (EC 2.4.1.691) that normally generates the H blood group locus and that may compete for the same substrate as the Gal3T. The reduction reported in cell surface α1–3Gal residues was further enhanced when α1–2 fucosyltransferase transgenic mice were bred to be cotransgenic with a transgene encoding an α-galactosidase that cleaves α1–3Gal linkages.

Transgenesis by pronuclear injection is a dominant genetic approach on a genomic background already containing a complete set of genes, as well as loci possibly mutagenized by transgene integration. In general, dominant transgenic approaches require reproducible, specific, and relatively high expression levels of the transgene, sometimes in a long-term manner, and this is not easily achieved. Although relevant physiologic information is still to be gained, a dominant genetic approach can complicate the assignment of structure-function relationships, and the random nature of transgene integration adds further complications.

Gene Targeting Using Embryonic Stem Cells ^(18–28)

The development of recessive genetic techniques in the mouse has provided a needed approach to understand the molecular genetic basis of physiological systems. Gene-targeting techniques are now routinely used to alter germ-line DNA by homologous recombination and therefore in a highly specific and experimentally defined manner. Homologous recombination between exogenous DNA and chromosomal DNA occurs at high efficiency in yeast and has been used with great success to produce recessive genetic lesions and thereby reveal the function of endogenous yeast genes. However, homologous recombination is a low-frequency event in the mammalian genome. Detection of this event using PCR has enabled the clonal selection of cells that have undergone homologous recombination in vitro following gene transfer. Coupling a PCR detection system with use of ES cells provided a major breakthrough in the late 1980s with the insertion of an experimentally altered allele into the mouse germ line.

ES cells are derived from the inner cell mass of blastocyst-stage embryos and can be maintained in vitro indefinitely in the pluripotent state with appropriate medium. This medium must be enriched in a cytokine originally termed leukemia inhibitory factor and now known by various trade names. Gene transfer into ES cells by electroporation is easily accomplished with stably transfected clones usually bearing single integration events that incorporate the neomycin phosphotransferase gene. Stable integrants are thus often selected by resistance to the antibiotic G418. The targeting vector is designed such that homology with genomic DNA is placed flanking the mutation to be generated (Figure 33.4). PCR screening and clone isolation follow, and subsequent studies at the genomic level following clone outgrowth can confirm the altered allelic structure. The frequency of homologous recombination, which may range from 0.1% to sometimes 10%, has been found to increase dramatically with the use of isogenic DNA, i.e., DNA from the same ES cell genome or strain of mice. Most ES cells have been derived from the 129 strain of mice, although ES cells from other strains, such as C57BL/6, have now been isolated.

Figure 33.4

Classic gene-targeting strategy used to inactivate an endogenous allele. The neomycin phosphotransferase gene (NEO) acts both as a selectable marker and as the insertional mutation that disrupts a coding exon of the gene.

Following the isolation and characterization of correctly targeted ES cells, they are microinjected into the “host” blastocyst-stage embryo (Figure 33.5). Approximately eight to ten ES cells are injected, and these integrate into the inner call mass, thereby colonizing various tissues in the developing embryo. If their colonization includes germ cells, the resulting mouse will transmit the altered allele to its offspring. The genotype of the host embryo is often C57BL/6 because this allows a coat color analysis, indicating the presence of chimeric mice born from 129-strain-derived ES cells (Figure 33.6). The 129 ES cell genome is homozygous for the Agouti locus and yields mice with brown coat color, whereas the C57BL/6 genome yields mice with black hair. However, the Agouti locus is dominant, and the amount of Agouti (brown hair) present on the chimeric mice coat tends to be an indicator of the likelihood that the ES cell genome has also colonized the germ cells. Breeding chimeric mice with C57BL/6 mice is a test for this and can generate mice heterozygous for the mutation. Pure Agouti offspring produced by this mating indicates that the ES cell genome has colonized the germ line. Of Agouti offspring, 50% will be heterozygous for a mutation that is “unlinked” in meiotic recombination to the Agouti locus. Subsequently crossing mice heterozygous for the mutation produces offspring that are homozygous for the mutant gene (25% on average), unless the mutation is lethal in embryogenesis.

Figure 33.5

Microinjection of gene-targeted ES cells into a blastocyst-stage embryo in producing chimeric mice.

Figure 33.6

Chimeric mice generated by injection of ES cells into a host (C57BL/6) blastocyst-stage embryo. Different amounts of chimericism (Agouti coat color) are apparent.

In this manner, hundreds of specific gene mutations have now been engineered in the mouse germ line in studies that are deciphering the molecular genetic basis of physiology and disease. A significant proportion of these mutations are found to be highly disadvantageous to the embryo or offspring born, often yielding serious developmental defects and lethality.

Investigating Gene Function in Vivo by Conditional Mutagenesis ^(29–40)

The goal of developing conditional mutagenesis approaches in vivo demanded the use of a site-specific DNA recombination system. The Cre and Flp recombination systems are intrinsic to bacteriophage and yeast, respectively, and thus have been used for this purpose. Both Cre and Flp are members of the integrase family of recombinases and can function without the use of ATP or cellular cofactors. They bind to 34-bp DNA sequences termed loxP for Cre or frt for Flp. Their mechanism of action is termed conservative and requires a Holliday intermediate structure with specific nucleotide base pairing at the recombination synapse. Cre recombinase appears to function better than Flp in mammalian cells, although development of more effective forms of Flp recombinase has recently yielded promising results. Cre can act in an efficient, heritable, tissue- and site-specific manner to excise DNA specifically flanked by direct repeats of loxP at distinct chromosomal locations. Following recombination, excised DNA is degraded, as it does not appear integrated elsewhere in the genome.

A novel mutagenesis strategy has been devised in which loxP sites are used in acquiring gene-targeted ES cell clones. Homologous recombination results in the incorporation of loxP sites flanking the gene element to be deleted (often a crucial exon) and the selectable markers, such as Neo and HSV-TK. Following transient Cre expression in these parental gene-targeted ES cells, subclones bearing either type I or type II recombination are selected for by the absence of the HSV-TK gene using gancyclovir (Figure 33.7). The type I recombinant (one loxP site left) is used to produce the systemic (classic) congenital deficiency. An advantage is the lack of prokaryotic Neo and HSV-TK genes, which can influence the expression of neighboring genetic loci and male fertility. The type II recombinant (two loxP sites remain) bears the conditional mutation, with loxP sites flanking the gene in what is expected to be a nondeleterious manner, from ES cell clones containing the conditional mutation. Following the production of chimeric and heterozygous mice, a breeding strategy to achieve conditional gene mutation in vivo is employed using transgenic mice bearing the Cre transgene (Figure 33.8). Mice homozygous for the floxed allele and transgenic for Cre will have tissues that undergo gene mutation only where Cre is expressed. Cre is provided as a transgene under the control of specific promoter-enhancer elements to generate a restricted expression pattern.

Figure 33.7

Strategy for achieving systemic and conditional gene mutagenesis in ES cells using the Cre-loxP recombination system.

Figure 33.8

Site-directed recombination and conditional gene mutagenesis in vivo. Mice bearing a tissue-specific Cre recombinase transgene expression profile in somatic cells (circled) are mated in generation 1 (G1) with mice heterozygous for a gene exon (boxed) (more...)

In practice, Cre recombination has been successfully demonstrated in vivo among virtually all somatic cell types, including postmitotic cells in the brain and liver. The efficacy is high in systems that produce high levels of nuclear-localized Cre protein, and chimeric tissues in which some cells have not undergone recombination also occur. The use of inducible promoters such as the tetracycline system and modification to the Cre sequence that allows hormonal activation are additional refinements that link Cre recombination to exogenous and experimental stimulation. Conditional mutagenesis in vivo obviously provides a more defined physiologic context in which to study gene function and may in some cases be necessary when systemic gene ablation leads to early lethality, thereby precluding the investigation of gene function in adult systems. The use of conditional mutagenesis by site-directed recombination is likely to be especially applicable in glycobiology where in vivo studies of glycans can require a focus on cell-cell interactions among relatively disparate cell types in an intact organism.

Functions of Glycans Revealed by Recessive Genetic Lesions ^(31,41–61)

To determine the function of glycans in vivo, enzymes participating in glycan diversification are now routinely inactivated in the mouse germ line. Using this recessive genetic approach, such mutations effectively restrict the formation of specific oligosaccharide linkages. When this approach is coupled to a defined biosynthetic and diversification pathway, like that of N-glycans, it is possible to define specific structure-function relationships of glycans in the intact animal. Like mutations generated in other obviously hierarchical biological systems, such as in protein phosphorylation, the inactivation of an enzyme can affect the structure of many “downstream” molecules. Very few to more than a dozen glycoproteins or phosphoproteins can be altered in systems deficient in a single enzyme. Nevertheless, such experiments are considered to represent the best approach currently available to establish the functions of genes, especially those that are conserved in phylogeny. As additional data are accumulated, it remains worth considering that perhaps not all posttranscriptional modifications are physiologically relevant. Overlapping functions have been found to exist, however, and some enzyme families and physiologic systems may thus have the ability to compensate for single gene dysfunction during ontogeny.

More than 20 genes controlling glycan biosynthesis and diversification have been inactivated in the mouse germ line as of this writing (Table 33.1). Studies have uncovered various and unexpected physiologic functions controlled by endogenous oligosaccharide structures. The phenotypes include embryonic and postnatal lethalities, widespread systemic disease with models of human glycosylation deficiencies, cell death by apoptosis, immune dysfunction, and defects in organogenesis. The lethalities observed thus far segregate to early steps in N-glycan biosynthesis, GPI-anchor formation, hyaluronan biosynthesis, and proteoglycan sulfation. In 26 studies that produced an inherited systemic glycan deficiency by gene targeting, only 5 (19%) caused embryonic lethality.

Table 33.1

Induced congenital oligosaccharide deficiencies in the mouse and their biological effects.

Most effects of glycan deficiencies thus far produced are evident in specific physiological systems and do not grossly alter morphogenesis or create systemic pathologies. This may reflect the experimental focus and assays performed. Cells commonly affected include those of the hematopoietic and nervous systems in processes that modulate hematopoiesis, immune function, receptor-ligand activation, myeloid inflammation, lymphoid apoptosis, neural axon migration, neuromuscular activity, endocrine function, and innervation. Additionally, lethal genetic deficiencies in early N-glycan biosynthesis and proteoglycan sulfation were detrimental to the development of the heart, kidney, neural tube, and vasculature. The modeling and study of vertebrate disease states such as the carbohydrate-deficient glycoprotein syndrome type II and the chronic glomerulonephritis induced by α-mannosidase II deficiency should provide information on the etiology of these glycan-based maladies.

In general, the more proximal the defect in glycan biosynthesis, the more widespread and severe the phenotypic outcome. This makes intuitive sense as the earliest steps in glycan diversification are common to most glycans and occur widely in many cell types. However, several early biosynthetic steps previously believed to be performed by a single enzyme have been found to involve the presence of numerous isozymes in vivo. In the case of α-mannosidase II deficiency, its role in early N-glycan biosynthesis remains as previously defined; however, its in vivo function was essential only to the erythroid lineage in the mouse. A distinct α-mannosidase activity was noted in Golgi extracts that could provide an efficient alternate pathway in complex N-glycan biosynthesis among most cell types (Chapter 7). A second Core 2 GlcNAcT isozyme has also been discovered (Chapter 8). Additional biosynthetic pathways operating in specific cell types may be more frequent than presently defined and may be further discovered from studies of in vivo recessive lesions among glycosyltransferase and glycosidase genes.

Several branchpoints exist in both N- and O-glycan biosynthesis (Chapters 7 and 8). Glycosyltransferases and glycosidases operating at these branchpoints control the formation of various structures in subsequent biosynthesis (see Chapter 16). Some of these structures are found on multiple branches, whereas others are preferentially localized to one branch. These branches may exist in part to create a multivalent ligand for the appropriate lectin. In studies ablating enzymes controlling branch formation, results so far generally indicate that N- and O-glycan branching provides unique functions that can be especially crucial for specific tissues or cell types (Figure 33.9). Studies inactivating other glycan classes in the mouse, including the biosynthesis of hyaluronan, GPI anchors, and glycosaminoglycan sulfation, have also provided a first glimpse of structure-function relationships involving these classes (Figure 33.10).

Figure 33.9

Branch specificity is observed in N- and O-glycan structure-function relationships. Circled structures represent those rendered deficient from induced germ-line mutations in the mouse involving indicated genes/enzymes that control N- or O-glycan diversification. (more...)

Figure 33.10

Structure-function relationships in glycosaminoglycan, hyaluronan, and GPI-anchor biosynthesis. Circled structures represent those rendered deficient by induced germ-line mutations in the indicated genes/enzymes of the mouse.

In theory, studying glycan function should also be possible by in vivo mutagenesis of the genes encoding specific lectin activities. Few such studies have been reported, and of those accomplished, the results obtained may be complicated by the potentially distinct role of the peptide component, as well as by the possible functional overlap with other lectins of similar binding activities. Nevertheless, physiological connections between the functions of glycosyltransferases and glycosidases and those of endogenous lectins are expected to contribute significantly to our understanding of how glycans modulate embryonic and adult physiology.

Future Directions

Studies thus far indicate that extracellular glycans perform roles in regulating cell-cell adhesion and the activation of receptor complexes through endogenous lectin-ligand interactions. These activities suggest that many glycan functions may be described as nature's biological modifiers or rheostat. Glycans can modulate the development and function of physiologic systems, as exemplified by the role of sialyltransferases in lymphoid apoptosis and antigen receptor signaling, and by the essential requirement for proteoglycans in fibroblast growth factor receptor activation. Endogenous lectins may therefore represent the “other arm” of glycan-based modulation systems and may be found to be more frequent and diverse among cells than currently recognized. In other situations, glycans may act primarily through steric and conformational influences. The finding of physiologic functions for genes controlling glycan formation provides a route to defining the mechanisms of glycan function. It will be of importance to develop new approaches to structural studies of glycans and their carriers as derived from whole tissues and primary cells of transgenic and gene-targeted mice. In understanding the physiologic processes that glycans modulate, experiments with various multicellular organisms will also be informative, and in some cases, breeding together multiple mutant genetic loci may uncover the presence of overlapping roles as well as yet to be discovered functions.

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