A recessive deletion in the GlcNAc-1-phosphotransferase gene results in peri-implantation embryonic lethality

Kurt W.Marek, Inder K.Vijay2, Jamey D.Marth1

Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, 9500 Gilman Drive-0625, University of California San Diego, La Jolla, CA 92093, USA and 2Department of Animal and Avian Sciences, University of Maryland, College Park, MA 20742, USA

Received on February 10, 1999; revised on April 5, 1999; accepted on April 5, 1999

Formation of the dolichol oligosaccharide precursor is essential for the production of asparagine- (N-) linked oligosaccharides (N-glycans) in eukaryotic cells. The first step in precursor biosynthesis requires the enzyme UDP-GlcNAc: dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT). Without GPT activity, subsequent steps necessary in constructing the oligosaccharide precursor cannot occur. Inhibition of this biosynthetic step using tunicamycin, a GlcNAc analog, produces a deficiency in N-glycosylation in cell lines and embryonic lethality during preimplantation development in vitro, suggesting that N-glycan formation is essential in early embryogenesis. In exploring structure-function relationships among N-glycans, and since tunicamycin has various reported biochemical activities; we have generated a germline deletion in the mouse GPT gene. GPT mutant embryos were analyzed and the phenotypes obtained were compared with previous studies using tunicamycin. We find that embryos homozygous for a deletion in the GPT gene complete preimplantation development and also implant in the uterine epithelium, but die shortly thereafter between days 4-5 postfertilization with cell degeneration apparent among both embryonic and extraembryonic cell types. Of cells derived from these early embryos, neither trophoblast nor embryonic endodermal lineages are able to survive in culture in vitro. These results indicate that GPT function is essential in early embryogenesis and suggest that N-glycosylation is needed for the viability of cells comprising the peri-implantation stage embryo.

Key words: mouse/GPT gene/peri-implantation/embryogenesis

INTRODUCTION

The posttranslational modification of proteins with asparagine (N)-linked oligosaccharides is a universal feature of eukaryotic cells. The earliest steps in N-glycan formation are conserved from yeast to humans with production of the dolichol oligosaccharide precursor. This structure is synthesized in a stepwise manner beginning with the enzyme UDP-GlcNAc: dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT). The function of GPT is to catalyze the transfer of GlcNAc-1-P from UDP-GlcNAc to dolichol-phosphate (Dol-P) to form GlcNAc-P-P-Dol and is necessary for subsequent steps required in generating the dolichol oligosaccharide precursor. The GPT gene is highly conserved among various eukaryotic organisms (Scocca and Krag, 1990; Zhu and Lehrman, 1990; Rajput et al., 1992) to the extent that the human gene encoding GPT can be isolated by complementation of the yeast homologue ALG7 (Eckert et al., 1998). GPT RNA expression in the mouse is widespread among adult cell types (Figure 1) and RNA levels vary during development and as a result of hormonal influences (Rajput et al., 1994a). Production and linkage of the dolichol oligosaccharide to nascent asparagine residues in the endoplasmic reticulum is followed by several processing reactions. The first few processing events are involved in protein folding mechanisms through recognition of terminal glucose residues by cellular chaperone lectins such as calnexin (Ou et al., 1993; Hammond et al., 1994; Hebert et al., 1995; Labriola et al., 1995). Subsequent diversification of N-linked oligosaccharide structures in the Golgi generates a cell surface and secreted N-glycan repertoire that can regulate cell adhesion, and signal transduction processes.


Fig. 1. GPT RNA expression among adult mouse tissues. Northern blot analysis using 5 µg of total RNA reveals that GPT is expressed in all tissues tested, although at varying levels. An analysis in parallel with half the RNA sample, and stained with ethidium bromide, is shown below to demonstrate comparable RNA loading.

In studies of mouse embryos, the specific activity of enzymes that mediate N-glycan formation have been found to decrease abruptly after fertilization but then increase during development of the blastocyst stage embryo (Armant et al., 1986). A requirement for N-glycans in early embryogenesis has been indicated in studies of embryos cultured with the antibiotic and antiviral compound tunicamycin, a GlcNAc analog that acts as a competitive inhibitor of GPT in the biosynthesis of the dolichol oligosaccharide precursor (Takasuki and Tamura, 1971; Bettinger and Young, 1975; Lehle and Tanner, 1976). Exposure to tunicamycin has been found to prevent gastrulation in sea urchin embryos and blocks pre-implantation development in mouse embryos cultured in vitro. Depending on the dosage and the stage at which treatment begins, tunicamycin can interfere with compaction, blastocyst formation, blastocyst hatching, and the growth of trophoblast cells (Surani, 1979; Atienza-Samols et al., 1980). However, the outcome following tunicamycin exposure has been inconsistent on occasion. This may reflect the specific cell types exposed, dosages, time of application, duration of exposure, preparations containing protein synthesis inhibitors, or additional mechanisms of tunicamycin action. Moreover, other oligosaccharide classes that incorporate GlcNAc during their biosynthesis, such as proteoglycans and glycolipids, may also be affected by tunicamycin (Eckardt et al., 1980; Stevens et al., 1982; Lohmander et al., 1983; Yanagishita, 1986).

Studies in mice genetically deficient in the Mgat1-encoded GlcNAcT-I enzyme have revealed that high-mannose type N-glycans are insufficient for development subsequent to embryonic day (E) 9 (Ioffe and Stanley, 1994; Metzler et al., 1994). The formation of N-glycans and initial processing events prior to embryonic Mgat1 function are therefore likely to be crucial in earlier developmental stages and ontogenic processes. In establishing structure-function relationships among vertebrate N-glycans in ontogeny, we have used the Cre/loxP recombination system to generate mice bearing either a conditional or systemic deletion in the gene encoding GPT. We find that an intact GPT gene is needed early in ontogeny and infer a role for N-glycosylation in peri-implantation embryonic cell viability.

RESULTS

GPT RNA expression is widespread in adult tissues

Total RNA isolated from various adult mouse tissues was subjected to Northern blot analysis using a full-length GPT cDNA probe. A transcript of the expected size at ~2.0 kb was found to be present in all tissues examined (Figure 1). Widespread expression of GPT is consistent with the ubiquitous presence of N-glycans among cell and tissue types in vivo.

Mutagenesis of the GPT gene in embryonic stem cells and the mouse germline

GPT is a transmembrane protein likely consisting of multiple membrane-spanning regions (Scocca and Krag, 1990; Zhu and Lehrman, 1990; Rajput et al., 1992) and is encoded by nine exons in the mouse genome (Rajput et al., 1994b). Exons 2 and 5 contain the two putative dolichol recognition sites (PDRS) both of which have been shown to be required for GPT function (Datta and Lehrman, 1993). We isolated a 15 kb mouse genomic clone by hybridization to GPT cDNA sequence which spanned the entire GPT-encoding region (Figure 2A). Sequence analysis revealed that deletion of the first two GPT exons would result in elimination of the transcription start site, the translation start site, as well as the first PDRS element, and therefore results in a null allele. A GPT gene targeting vector was thus constructed to enable this excision using Cre-loxP mutagenesis (Figure 2B; reviewed by Marth, 1996). Embryonic stem cell (ES cell) clones which were resistant to G418 were isolated and analyzed for homologous recombination by PCR and Southern blotting (Figure 2C, left panel). Homologous recombinants that contained three loxP sites (Figure 2C, right panel) were subjected to transient Cre recombinase expression. ES cell subclones bearing systemic (clone 7.2.5, 7.2.10) and conditional (clones 7.2.7, 7.2.8) mutations in the GPT gene were thereby isolated.


Fig. 2. GPT mutagenesis in ES cells and the mouse germline. (A) A mouse genomic clone containing all nine exons of the GPT gene was used with the pflox vector to generate the targeting vector (numbered black boxes represent exons, the UTR regions are shaded). The GPTF[tk-neo] allele is produced after homologous recombination of targeting vector in the R1 ES cells. Restriction enzymes: N, NotI; B, BamHI; R, EcoRI; Bg, BglII; K, KpnI; S, SpeI. The BglII site that is shown with an asterisk is polymorphic and only found in the C57BL/6 strain. (B) ES cells with the GPTF[tk-ne°] allelic structure were exposed to transient Cre expression and ganciclovir selection to generate systemic (GPT[Delta]) and conditional (GPTF) mutations by deletion of loxP-flanked DNA. (C) Southern blotting was used to confirm genomic GPT allelic structures. Left panel: clone 7.2 is a homologous recombinant bearing both the targeted (F[tk-neo]) and wild type (wt) GPT alleles. Right panel: clone 7.2 contains three loxP sites and was used to generate ES cell clones containing the "[Delta]" (7.2.5, 7.2.10) and "F" (7.2.7, 7.2.8) GPT alleles. (D) Left panel: tail DNAs from offspring of heterozygous parents (wt/[Delta]×wt/[Delta] were analyzed by Southern blotting. Only wild type (wt/wt) and heterozygous (wt/[Delta]) mice were present. Right panel: mice heterozygous for the conditional mutation (GPTwt/F) were crossed and tail DNA of the offspring was analyzed by Southern blotting. Offspring homozygous for the GPTF allele were observed at normal frequency. A polymorphic BglII site (A, Bg*) is present in the wild-type GPT allele in the C57BL/6 background.

Chimeric mice were generated from clones 7.2.5 and 7.2.7 and were bred to C57BL/6 females to produce mice heterozygous for the deleted (GPT[Delta]) and conditional (GPTF) alleles. GPTwt/[Delta] mice were crossed and offspring with GPTwt/wt and GPTwt/[Delta] genotypes were obtained at a 1:2 ratio, respectively; however, no GPT[Delta]/[Delta] offspring were found in over 200 screened (Figure 2D, left panel; and data not shown). These results indicate that the deletion generated in the GPT gene is a recessive lethal lesion in the mouse germline. Mice heterozygous for the conditional mutation (GPTwt/F) were crossed and offspring homozygous for the conditional allele (GPT F/F) were found among ~25% of offspring (Figure 2D, right panel; and data not shown). These GPTF/F animals were normal and fertile.

Cells heterozygous for the GPT deletion exhibit a variable decrease in GPT enzyme activity

To determine if the deletion in the GPT gene results in a deficiency in GPT activity, an analysis of GPT activity was performed on embryonic stem cells, and on the liver and mammary glands of 12 day lactating mice heterozygous for the GPT gene deletion. The specific activity of GPT can be determined by measuring the transfer of UDP-GlcNAc to dolichol phosphate as described previously (Shailubhai et al., 1988). GPT activities in GPTwt/[Delta] ES cells and the liver from GPTwt/[Delta] mice were found to be ~50% that of wild-type samples (Figure 3). While the average activity level was slightly lower in the mammary gland, it was more similar to that observed in wild-type tissue (Figure 3B). This may reflect a lower turnover of GPT in this tissue, or perhaps the presence of a second GPT isozyme. Nevertheless, these data indicate that the introduced GPT gene deletion, which ablates the transcription start site, the translation start site, and a PDRS element, results in a defective allele providing little or no GPT enzyme activity. The absence of sufficient material from embryos homozygous for the GPT gene deletion precluded similar biochemical analyses in this mutant background.


Fig. 3. Reduced GPT activity in cells and tissues of mice heterozygous for the GPT gene deletion. ES cells (A) or livers and 12-day lactating mammary glands (B) from wild-type (black) or heterozygous (white) mutant mice were analyzed for GPT enzyme activity as described previously (Materials and methods). The mean specific activity of GPT is shown for three separate studies using ES cells and tissue samples. Standard deviations are denoted with error bars.

Embryos homozygous for the GPT mutation die in peri-implantation development

To determine when in embryogenesis homozygous GPT deletion caused lethality, GPTwt/[Delta] mice were bred and the females were sacrificed at various times postfertilization. The uteri were dissected and inspected for resorption sites, as detected by the presence of relatively small degenerating embryos (Baines et al., 1996). Resorption sites were apparent at E9.5, E8.5, and E7.5 among ~25% of decidua (Table I). At these times specific embryonic tissues were not identifiable, indicating that the embryos had died earlier in gestation. All uterine decidua at E6.5 and E5.5 appeared equivalent in size and were harvested for histologic examination. Approximately 25 percent of E6.5 decidua contained evidence of embryo death with resorption underway and an absence of viable embryonic cells.

Table I. Embryo resorption frequencies during postimplantation development
  Parental genotypes: wt/[Delta] and wt/[Delta] Parental genotype: wt/[Delta] and wt/wt
Embryonic day No. of decidua No. in resorption % Resorption No. of decidua No. in resorption % Resorption
8.5-9.5 27 7 26 8 0 0
7.5 24 6 25 9 0 0
6.5 14 4 29 ND ND ND
5.5 19 3 16 10 0 0
ND, Not determined.

A significant proportion of E5.5 embryos derived from heterozygous matings were beginning to undergo resorption with cellular degeneration of both extraembryonic and embryonic cell types (Figure 4). To determine the frequency of spontaneous resorptions in the hybrid 129×C57BL/6 strain used in these studies, female littermates that were homozygous wild-type for the GPT gene were mated to the heterozygous males used in the above studies. No resorption sites were observed in 27 concepti examined between E5.5 and E9.5 (Table I) indicating embryonic death was due to homozygosity for the GPT deletion.


Fig. 4. Recessive deletion in the GPT gene results in embryonic lethality by E5.5. Hematoxylin and eosin were used to stain 5 µm sections of paraffin-embedded E5.5 decidua obtained from a heterozygous GPT breeding (wt/[Delta]×wt/[Delta]). (A, B) Representative sections of the E5.5 embryo resorption sites analyzed. Embryonic tissue is present in a state of gross degeneration. (C) No defective embryos similar to those in (A) or (B) were observed when heterozygous (wt/[Delta]) mice were bred to wild-type (wt/wt) mates.

GPT-deficient blastocysts form abnormal and deficient blastocyst outgrowths in vitro

While embryos homozygous for the GPT deletion were inviable, it was uncertain whether cells derived from these embryos could be maintained in vitro and further analyzed. Blastocyst stage embryos, which comprise ~60 cells, were isolated from female mice heterozygous for the GPT deletion 3.5 days after fertilization by heterozygous males. The blastocysts were cultured separately and observed for 4 days. All E3.5 blastocysts were morphologically indistinguishable; however, after 1 day in culture ~25% of the embryos failed to hatch from the zona pellucida and began to collapse and die. Embryos that hatched were genotyped using the polymerase chain reaction and found to be either homozygous wild-type or heterozygous for the GPT deletion (see below). These embryos all generated morphologically normal blastocyst outgrowths with trophoblast giant cells surrounding a colony of inner cell mass (ICM) cells.

To determine if the hatching deficiency precluded embryonic cell survival in vitro, the zona pellucidae was chemically removed using acid tyrode solution (see Materials and methods). Unlike wild-type or heterozygous embryos, those homozygous for the GPT deletion did not generate trophoblast outgrowths upon subsequent culture (Figure 5). Trophoblast cells from GPT[Delta]/[Delta] embryos did not adhere to the plate but instead became rounded and degenerated by 48 h post-culture (Figure 5A). All trophoblast cells appeared dead by 72 h. Embryonic cells of the ICM of null embryos began to disaggregate and die after 48 h in culture. Embryo cultures failing to support trophoblast or ICM cell outgrowth were invariably those in which both GPT alleles were mutated (Figure 5B), using a strategy based upon the polymerase chain reaction to genotype the GPT alleles. Although a Mendelian ratio of genotypes was observed among blastocysts derived from heterozgyous parents, embryonic cells that lacked an intact GPT gene could not be maintained and cultured in vitro.


Fig. 5. GPT[Delta]/[Delta] blastocyst explants fail to survive in vitro. (A) Blastocysts at E3.5 were obtained from timed matings of mice bearing GPTwt/[Delta] genotypes and placed on gelatinized tissue culture plates in DMEM media. Among wild-type and heterozygous blastocysts (top two rows), the trophoblast giant cells spread out to form a monolayer while the inner-cell mass cells form a growing colony in the center. In embryos homozygous for the GPT deletion, the trophoblasts do not spread out on the plate and the inner-cell mass cells do not survive. Cell types and structures: t, trophoblasts; i, inner-cell mass; zp, zona pellucida. (B) At E3.5+96 h, outgrowths shown in A were lysed and genotyped by PCR. Lanes 1-3 denote the results obtained from embryos in the upper row, middle row, and lower row, respectively. These results were used to assign the GPT genotypes denoted in (A). The PCR genotyping strategy showing the location of the oligonucleotide primers is detailed on the right.

In assessing the presence of N-glycans using the lectins concanavalin A (ConA) and erythrophytohemagglutinin (E-PHA), all embryonic cells analyzed from E5.5 and earlier continued to bind lectins specific for N-glycans, regardless of the GPT genotype (data not shown). This may reflect the presence of a second GPT gene or a parental GPT source available to the zygote as was indicated in studies of embryos lacking a functional Mgat1 gene (Campbell et al., 1995; Ioffe et al., 1997). Our findings are similar to those observed obtained upon tunicamycin treatment of embryonic cells where N-glycans and low levels of GPT activity persisted prior to cell death (Surani, 1979; Atienza-Samols et al., 1980; Surani et al., 1981; Armant et al., 1986). Our results indicate that the GPT deletion engineered in the mouse germline is a recessive lethal mutation during postimplantation embryogenesis around E4.5-E5.5.

DISCUSSION

The structure of the dolichol oligosaccharide precursor found in all eukaryotic cells indicates that GlcNAc-1-phosphotransferse activity should be essential for precursor biosynthesis and thus for cellular N-glycosylation. Consistent with this view, tunicamycin has been found to inhibit both GPT activity and N-glycosylation in culture. However, tunicamycin may interfere with the formation of proteoglycans, glycolipids, and the palmitoylation of proteins (Eckardt et al., 1980; Stevens et al., 1982; Lohmander et al., 1983; Yanagishita, 1986; Patterson and Skene, 1994; Takahashi et al., 1997), suggesting the possibility of effects unrelated to a loss of N-glycan formation. For example, the plant alkaloid swainsonine inhibits [alpha]-mannosidase-II activity (Elbein et al., 1981) and has been used to study complex N-glycan biosynthesis and function. However, swainsonine is also known to inhibit lysosomal [alpha]-mannosidase activity (Dorling et al., 1980) and this may account for phenotypic differences observed in comparing swainsonine administration with a genetic deficiency induced in the [alpha]-mannosidase-II gene (Chui et al., 1997). Since some of the phenotypic effects following tunicamycin treatment might not be due to inhibition of GPT, an approach to explore this possibility required an inactivation of the gene encoding GPT with a comparison of the resulting embryonic phenotype to that reported with tunicamycin treatment. In so doing we have found similarities and some differences between the effects of tunicamycin and GPT deficiency in the early stages of embryo development.

All embryos lacking an intact GPT gene were found to develop normally to the morula and blastocyst stages in vitro, unlike results obtained using moderate to high doses of tunicamycin which caused developmental arrest and decompacted blastomeres of early morula stage embryos (Surani, 1979; Surani et al., 1981). This difference may reflect a maternal source of GPT protein in mice homozygous for the deletion in the embryonic GPT gene (and see below) or perhaps a second GPT isozyme with an early embryonic expression pattern. In earlier studies using tunicamycin, ICM cells were found to be less sensitive to moderate doses and survived after trophoblast death (Atienza-Samols et al., 1980), although decreased viability of embryonic cells was noted with increased tunicamycin dosages (Surani, 1979). We find that GPT deficiency completely inhibits trophoblast adhesion and outgrowth and both trophoblast and ICM cells die shortly after explant culture in vitro.

Similar to results reported by Surani and others, we find that blastocysts are frequently unable to hatch from the zona pellucida (Atienza-Samols et al., 1980). This effect could result in a defect in embryo implantation. However, this does not appear to be the case in vivo as 25% of embryos inducing a decidual reaction in the uterine epithelium, and found undergoing resorption between E5.5 and E9.5, were derived specifically from heterozygous GPTwt/[Delta] parents (Table I), indicating that hatching and implantation occur without embryonic GPT gene function. Why hatching would occur in vivo but not efficiently in vitro is unclear, although embryos in vivo may be subject to more mechanical stress that might promote the hatching process. While neither GPT deficiency nor tunicamycin treatment of embryos results in an absence of N-glycans, there is similarity among the preimplantation embryonic phenotypes observed. Nevertheless, in resolving the issue of whether N-glycans are required in early embryogenesis, new experimental approaches must be developed.

During the course of these studies we have generated a conditional GPT mutation (GPTF) that can be bred to various Cre transgenic backgrounds (Orban et al., 1992; Marth, 1996). This will facilitate tissue-specific and temporal studies of GPT, and other enzymes such as Mgat1-encoded GlcNAcT-I, that are suspected to be playing an unique roles in specific cell lineages, as well as in pre-implantation development. For example, mice have been derived that express the Cre recombinase specifically in oocytes under the control of the zona pellucida 3 (Zp3) promoter prior to the final meiosis in oocyte development (Philpott et al., 1987; Lewandoski et al., 1997). By mating female GPTF/[Delta] mice that also carry the Zp3-Cre transgene with GPTF/[Delta] males, it is possible to generate GPT[Delta]/[Delta] embryos that may contain little or no maternal source of GPT. Conditional mutagenesis using the Cre recombinase may also be useful in deriving various cell-type-specific lines that can be induced to become genetically null in vitro using various Cre expression systems. Such conditional-mutant systems may be needed to provide sufficient material for biochemical studies of N-glycan formation during an elimination of essential gene function.

A deficit in N-glycosylation can lead to abnormal protein folding with loss of bioactivity as well as reduced trafficking to the cell surface, thereby precluding the generation of molecular interactions that promote cell survival and proliferation. While not all cell surface and secreted glycoproteins bearing N-linked oligosaccharides may require their attached N-glycans for expression and function, a significant number apparently do. It has been found that tunicamycin interferes with the production of glutamate receptors, Na+ channels, lipoprotein lipase, the T lymphocyte CD4 glycoprotein, immunoglobulins IgA and IgE, thrombin receptors, transferrin receptors, erythropoietin, and platelet-derived growth factor receptor-induced expression of insulin-like growth factor 1 (Hickman et al., 1977; Konig et al., 1988; Reckhow and Enns, 1988; Sumikawa et al., 1988; Masuno et al., 1991; Musshof et al., 1992; Kitagawa et al., 1994; Tordai et al., 1995; Carlberg and Larsson, 1996).

We did not observe any phenotypic or pathologic effect among mice or tissues heterozygous for the GPT gene deletion. Therefore, inactivation of a single GPT allele does not result in defects characteristic of the type I Carbohydrate Deficient Glycoprotein Syndromes which are linked to reduced N-glycosylation (Jaeken et al., 1997). However, it is possible that primary somatic cell types are differentially affected by a partial reduction in N-glycan formation. This may occur from different growth factor requirements and stimulus response programs that involve N-glycans on distinct glycoproteins. Conditional mutagenesis in intact animals and primary somatic cells bearing a GPTF/F genotype will be a valuable approach in exploring this possibility. Mutagenesis in germ cells on the other hand has a provided a congenital defect revealing a requirement for an intact GPT gene in embryogenesis with the implication that N-glycosylation is crucial for early embryonic cell viability.

MATERIALS AND METHODS

Northern blot analysis

Total RNA was prepared from various mouse tissues, electrophoresed on a 1% formaldehyde gel, and processed for hybridization as described previously (Priatel et al., 1997). The blot was probed with a mouse GPT cDNA bearing the complete coding sequence.

Generation of targeted ES cells and GPT deficient mice

A 15 kb clone containing the entire GPT gene was isolated by probing a mouse 129/SvJ genomic library (Stratagene) with a GPT genomic probe (Rajput et al., 1994b) containing the first three exons. A gene-targeting vector was generated by cloning a 1.2 kb NheI-EcoRI genomic fragment containing the first two exons into the BamHI site of pflox (Figure 2A). Flanking regions of 4.2kb (NotI-NheI) and 1.0 kb (EcoRI-SpeI) were inserted into the HindIII and XbaI sites, respectively, of pflox to complete the targeting vector. The targeting vector was linearized using NotI, purified using the Gene-Clean Kit (Bio 101), and 10 µg of DNA was electroporated into R1 ES cells. Electroporated ES cells were cultured for 7-10 days in media containing G418 (120 µg/ml, Life Technologies). Colonies were isolated and screened for homologous recombinants by PCR using thymidine kinase promoter (5[prime]-TGC AAA ACC ACA CTG CTC GAT CCG-3[prime]) and GPT specific (5[prime]-AGG TTG AAG ACA ATG ATA GAA G-3[prime]) oligonucleotide primers. Homologous recombinants (~1/150 G418-resistant clones) were confirmed by Southern blot analysis using a BglII digest and a 271 bp KpnI-DraI cDNA probe specific for exons 6 and 7. In addition, a loxP probe generated from a 192 bp NotI fragment of pLox2 (Orban et al., 1992) was used with a BamHI/BglII digest to confirm the presence of three loxP sites. Clone 7.2 was confirmed to be a homologous recombinant containing three loxP sites and was electroporated with 1.5 µg of plasmid encoding the Cre gene; 0.5 µm ganciclovir was added to the media after 3 days and clones were isolated after another 5 days in culture. ES cell clones heterozygous for the deletion ([Delta]) (7.2.5, 7.2.10) and the conditional (F) mutation (7.2.7, 7.2.8) were used to generate chimeric mice by blastocyst injection. Chimeric male mice were bred to C57BL/6 females to generate mice bearing either the null or conditional mutation and the mutations were maintained on the C57BL/6 background for at least four generations.

Analysis of GPT enzyme activity

GPT enzyme assays were performed in triplicate on ES cells and mouse tissues as described previously (Vijay et al., 1980; Shailubhai et al., 1988).

Histology

Uteri were removed from E5.5 and E6.5 pregnant females and fixed overnight in 4% paraformaldehyde at 4°C. The uteri were cut between each decidua and then processed by dehydrating through a graded ethanol series and clearing with Histo-Clear (Fisher Scientific) for 10 min at each step and subsequently embedded in paraffin. 5µm serial sections were cut on a microtome and stained with hematoxylin and eosin.

In vitro culture of blastocyst outgrowths and genotyping by polymerase chain reaction

Blastocysts were cultured as previously described (Liu et al., 1996). Briefly, heterozygous timed matings were set up and females were sacrificed 3.5 days later. The uteri were removed and flushed with blastocyst media (DMEM, 20% fetal calf serum, BSA (4 mg/ml), 2 mM L-glutamine, penicillin-streptomycin, and 0.1 mM 2-mercaptoethanol). Blastocysts were segregated into individual wells of a gelatinized 96-well plate and cultured in blastocyst media for 4 days. The zona pellucidae were removed after 16 h in culture by briefly exposing the blastocysts to acid tyrode as described previously (Hogan et al., 1986). Genotyping was performed by lysing the outgrowths for 1 h at 55°C in 30 µl of lysis buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2 mM MgCl2, and Proteinase K (10 µg). The samples were heated to 95°C for 10 min and then added to a PCR reaction containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2 mM MgCl2, and three oligonucleotide primers. The antisense primer e3 (5[prime]-GATTGAGGACATCATCAGCAAAC-3[prime]) is common to both the wild type and null alleles. The sense primer e2 (5[prime]-CCAGAGTCCCAGGGAGTGATC-3[prime]) was used to detect the wild type allele by generating a 450 bp product and the sense primer l1 (5[prime]-CCCTCGACCTGCAGCCCAAG-3[prime]) was used to detect the null allele by generating a 150 bp product.

ACKNOWLEDGMENTS

This research was supported by N.I.H. Grants DK48247 to J.M and DK19682 to IKV. J.M. is an Investigator of the Howard Hughes Medical Institute.

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1To whom correspondence should be addressed at: Howard Hughes Medical Institute, 9500 Gilman Drive-0625, University of California San Diego, La Jolla, CA 92093


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