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Proc Natl Acad Sci U S A. Sep 2, 1997; 94(18): 9920–9925.
PMCID: PMC23295
Neurobiology

Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos

Abstract

Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes—survival motor neuron (SMN) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the SMN gene product in vivo we generated SMN-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one SMN gene. Mice with homozygous SMN disruption display massive cell death during early embryonic development, indicating that the SMN gene product is necessary for cellular survival and function.

Proximal spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by loss of spinal motor neurons. The degeneration of these neurons leads to predominantly proximal symmetric muscle weakness and atrophy (13). Several types of the disease are distinguished by their time course and degree of motor function loss during postnatal development (4): In type I, the disease is manifest within the first 6 months of life; affected children generally die within the first 4 years of life. Type II patients show clinical weakness before 18 months and never stand or walk without support. Type III patients become symptomatic between 18 months and 30 years of life, with variable degrees of proximal weakness.

While this clinical classification system provides useful prognostic clues (5), molecular analysis has shown that both severe early onset and mild late onset forms are linked to the same chromosome locus 5q13 suggesting genetic homogeneity (69). Positional cloning strategies and deletion analysis have led to the identification of three candidate genes for spinal muscular atrophy, survival motor neuron SMN (10), neuronal apoptosis inhibitory protein NAIP (11), and transcript XS2G3 (12) [corresponding to exon 7 of the NAIP gene in reverse orientation (13)]. All these candidate genes are positioned within a complex region that is duplicated on the long arm of chromosome 5 (14), resulting in two copies of the NAIP and the SMN gene in the human genome. The NAIP gene shows homology to two baculovirus genes encoding inhibitor of apoptosis proteins (11). Its forced expression in vitro has been shown to suppress apoptosis in nonneuronal mammalian cells (15). NAIP exon 5 is deleted in 60% of type I SMA patients; in milder forms of the disease, deletions are identified only in 18% of affected individuals (11, 1618). This gene is also deleted in 3% of normal carriers (11, 17), arguing that it is not essential for motor neuron survival or that its function can be compensated by other genes, in particular the truncated centromeric copy.

In contrast, 95% of SMA patients have deletions of the telomeric SMN gene regardless of phenotype severity (10, 1620). This has led to the assumption that the SMN gene is the more likely candidate whose dysfunction is responsible for the disease (1618). The two copies on chromosome 5q13, telomeric SMN copy (SMNtel) and centromeric SMN copy (SMNcentr), differ only in five nucleotides in their coding regions, none of which causes an amino acid change. Rarely, asymptomatic carriers also show homozygous deletion of SMNtel (16, 17, 20, 21). SMNcentr is deleted in 2–3% of normal carriers (10, 1719). Four different small intragenic mutations have been identified in SMA patients that specifically disrupt SMNtel, providing further evidence for the hypothesis that SMNtel is the SMA disease-causing gene (10, 22). The centromeric gene copy (SMNcentr) is also expressed (10). However, developmental expression patterns and tissue distribution of these two isoforms have not been determined so far. Thus, it remains to be shown whether SMNcentr could compensate in case of the deletion of the telomeric copy or whether differential expression of both genes would not allow such functional overlap.

The SMN gene shows no homology to previously identified genes. Recently, a search for binding partners of the heterogeneous nuclear ribonucleoprotein U has identified SMN and subsequent immunohistochemical analysis revealed SMN localization in specific nuclear structures called gems (23). Based on the association of gems with coiled bodies a role of SMN in RNA metabolism has been suggested. Because the SMN gene is widely expressed both in neuronal and nonneuronal tissue (10), the specific degeneration of motor neurons, which is characteristic for spinal muscular atrophy, remains to be explained.

To characterize the role of the SMN gene during mammalian development we have identified the SMN homolog in the mouse and generated mice in which this gene is disrupted. In contrast to the human gene SMN is a single copy gene in the mouse. SMN-deficient mouse embryos develop normally until the compacted morula stage. Subsequent morphological alterations and degenerative changes lead to embryonic death prior to uterine implantation. The presence of degenerating, terminal deoxynucleotidyltransferase-mediated UTP endlabeling (TUNEL) positive cells in SMN−/− embryos demonstrates an essential role of the SMN protein for cellular survival and function and its potential involvement in regulatory processes underlying programmed cell death.

MATERIALS AND METHODS

Identification of the Mouse SMN Gene.

A 527-bp PCR fragment corresponding to nucleotides 329–855 of human SMN (10) was used to screen a newborn mouse brain λZAP cDNA library (Stratagene) at low stringency. One candidate clone with an insert length of 1.3 kb (pSC4) contained an 891-bp ORF with high homology to the human SMN gene (exons 1–6). The C-terminal coding region corresponding to human exon 7 was identified by reverse transcription–PCR to be homologous as well. This region was separated from exon 6 by an unspliced intron of 106 bp in the pSC4 clone.

Generation of SMN Knockout Mice.

Using pSC4 as a probe, two overlapping genomic clones, pSC6 and pSC7, were identified from a genomic library of 129/Ola-derived embryonic day (E) 14 embryonic stem (ES) cells, λPS (24). Using primers homologous to exons 1–6 of the human SMN gene in sequencing reactions, both clones were found to contain exons 2–4 and flanking intronic sequences. An 840-bp HindIII fragment of pSC7 containing the 5′ end of exon 2 was ligated into the XmnI site of the targeting vector pGNA (25). The 3.8-kb HindIII/KpnI fragment of pSC7 was then introduced into the SmaI/KpnI site of pGNA. The KpnI linearized targeting construct was electroporated into E14Tg2a-IV ES cells, grown without feeders and selected in the presence of G418 as described (26).

Homologous integration events were screened for by PCR and confirmed by restriction analysis of both 5′ (probe a) and 3′ ends (probe b) (Fig. (Fig.11b). Probe a was a 500-bp BamHI/HindIII fragment upstream of the targeting construct, probe b was a 330-bp fragment (aa 65–174) amplified by PCR from pSC4. Five of 125 ES cell clones showed homologous integration events. Two clones injected into C57BL/6 blastocysts gave rise to germ-line transmission of the mutated allele. Chimeras were mated with outbred MF1 albino females. Heterozygous offspring were backcrossed to the MF1 strain. Intercross progeny were generated at the N1 and N2 generations. Progeny and E12 embryos were genotyped by Southern hybridization of mouse tail DNA BamHI digests using probe a.

Figure 1
Disruption of the murine SMN gene. (a) Amino acid sequence of the human (10) and mouse SMN protein. Uppercase letters represent related residues, shaded boxes highlight identities. The protein appears highly conserved with 83% identical and 98% ...

Analysis of Embryos.

Following superovulation and mating of SMN+/− female mice with SMN+/− males the animals were sacrificed approximately 56 hr after mating and early morulae were obtained by tearing the oviducts. Each embryo was subsequently transferred to a drop of M16 medium overlaid with mineral oil and incubated at 37°C and 5% CO2/95% air. Under these conditions (27), wild-type embryos could be maintained for 5 days without morphological evidence of degeneration (data not shown).

Embryos were photographed after 24 and 36–48 hr of culture before they were harvested for DNA analysis. Each embryo was transferred into a PCR tube containing 10 μl of PCR lysis buffer (50 mM KCl/10 mM Tris, pH 8.3/2.5 mM MgCl2/0.1 mg/ml gelatin/0.45% Nonidet P-40/0.45% Tween 20/200 μg/ml Proteinase K) and incubated at 55°C for 1 hr and at 95°C for 15 min. Amplification of the mutant allele was performed with the primer pair used for the ES cell screen (forward 5′-CGTCTTATGGTATGGCAACTG-3′, reverse 5′-CATGCTGGGTACATGAAAACC-3′), amplification of the wild-type allele with a primer pair spanning the insertion site (sense 5′-GATGATTCTGACATTTGGGATG-3′, antisense 5′-TGTTTCAAGGGAGTTGTGGC-3′). PCR conditions were: a 1-min 95°C heating step, followed by 30 cycles of 95°C for 30 sec, 56°C for 1 min, 72°C for 90 sec, and a final 10 min incubation at 72°C. Samples were run on 2% agarose gels, blotted, and hybridized according to standard methods (28).

TUNEL Staining.

Embryos derived from SMN+/− intercrosses were washed with PBS and fixed in 4% paraformaldehyde/PBS for 5 min at room temperature. They were subsequently washed three times with PBS for 5 min and permeabilized in 0.1% Triton X-100/0.1% sodium citrate for 2 min on ice. Following two rinses with PBS they were incubated with terminal deoxynucleotidyl transferase and fluorescein-labeled dUTP (In Situ Cell Death Detection Kit, Boehringer Mannheim) at 37°C for 1 hr. Embryos were subsequently destained three times with PBS for 10–15 min before they were analyzed and photographed under the fluorescence microscope. Except for the labeling mix, 1% FCS was added to all solutions to prevent the fragile embryos from sticking to the surface of the plastic dishes. After analysis, the embryos were individually transferred into PCR tubes and processed for genotyping as described previously. To increase the sensitivity of this analysis, a multiplex PCR assay was applied using a common forward primer (5′-CTCCGGGATATTGGGATTG-3′) and two reverse primers specific for the wild-type (5′-GTTGTGGCATTCTTCTGGC-3′) and mutant (5′-GGTAACGCCAGGGTTTTCC-3′) SMN genes. PCR conditions were identical with those described above. This methodological change allowed us to increase the number of embryos that could be genotyped after TUNEL staining. Five embryos from three litters could thus be identified as homozygous mutants and all of them showed strong TUNEL staining as shown in Fig. Fig.22k.

Figure 2
Morphological alterations and TUNEL staining in control and mutant SMN embryos. (ae) Wild-type or heterozygous embryos. (fk) Homozygous mutant embryos at equivalent time points. A failure in transition to the blastocyst stage first became ...

LacZ Expression.

Whole-mount embryos were fixed in 2% paraformaldehyde/0.2% glutaraldehyde/PBS for 5 min, washed three times with PBS, and stained overnight at 37°C in 1 mg/ml 4-Cl-5-Br-3-indolyl-β-galactoside [4% 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal) dimethylsulfoxide stock solution], 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2 in PBS as described (29). Ovaries were immersion-fixed with 2% paraformaldehyde for 30 min, washed briefly with PBS, and cryoprotected in 30% sucrose/PBS overnight. The tissue was embedded in Tissue-Tek (Miles), frozen by immersion in isopentane, and mounted on gelatine-coated slides after cryostat sectioning. Following a brief rinse in PBS, sections were fixed with 0.2% glutaraldehyde, 5 mM EGTA, 2 mM MgCl2/PBS for 5 min, rinsed briefly, and stained with the X-Gal solution (see above) at 30°C overnight. After a rinse in PBS these sections were counterstained for 1 min with 1% neutral red in 50 mM sodium acetate buffer (pH 3.3) before dehydration in 70%, 90%, and 96% ethanol (modified from ref. 27).

Histological Analysis of Facial Motoneurons.

Five-month-old SMN+/+ and SMN+/− mice were perfused with 4% paraformaldehyde, and the brainstem regions containing the facial nuclei were dissected and processed as described (30). Briefly, paraffin serial sections (7 μm) were prepared and stained with cresyl violet. Only those facial motoneurons that showed a clearly detectable nucleus and nucleolus were counted in every fifth section. The motoneuron counts were corrected for double counting of split nucleoli, applying the formula introduced by Abercrombie as described (30).

RESULTS AND DISCUSSION

Cloning and Gene Targeting of the Mouse SMN Gene.

As a first step in our analysis, a mouse SMN cDNA clone was identified from a newborn mouse brain cDNA library by low stringency hybridization using a probe corresponding to nucleotides 329–855 of the human SMN cDNA sequence (10). The predicted mouse SMN protein shares 83% amino acid identity with the human protein (Fig. (Fig.11a). Northern blot analysis indicates that SMN mRNA is widely expressed (data not shown), which corresponds to the expression of SMN mRNA in human (10). In contrast to multiple transcripts of the human SMN genes originating from alternative splicing of exons 5 and 7 (10, 32), no evidence for alternative splicing in the mouse SMN sequence corresponding to the C-terminal part of the SMN protein was obtained. reverse transcription–PCR analysis of exons 4–8 revealed only a single amplification product from RNA derived from mouse embryonic stem cells, primary embryonic fibroblasts, brain, spinal cord, muscle, spleen, kidney, and testes (data not shown).

Restriction analysis of genomic mouse DNA indicated that the mouse genome contains only one copy of the SMN gene. This observation was corroborated by Southern blot analysis of ES cells after homologous recombination (see below). We designed a targeting vector in which the Escherichia coli lacZ gene is fused in-frame to the first 40 nucleotides of exon 2. A neomycin resistance gene under a separate promoter is positioned 3′ to the lacZ sequence (Fig. (Fig.11b). Homologous integration into the SMN locus in five independent ES cell clones was verified by Southern blot hybridization. The 1:1 ratio of signal intensity of bands corresponding to mutant and wild-type SMN in all five ES cell lines confirmed that SMN in the mouse is a single copy gene (data not shown). Two independent lines of heterozygous mice were generated following blastocyst injection.

Intercrosses of heterozygotes from both lines failed to produce homozygous SMN−/− progeny. Analysis of E12 embryos from heterozygous intercrosses also did not show any SMN−/− progeny (Table (Table1).1). Heterozygous intercrosses segregated SMN+/− and SMN+/+ in a 2:1 ratio, indicating that homozygous SMN disruption results in an early lethal phenotype (Fig. (Fig.11c). There was no indication of increased uterine resorption, suggesting that SMN−/− embryos do not implant.

Table 1
SMN+/− intercross progeny

Massive Cell Death in Early SMN−/−Embryos.

Progeny from heterozygous intercrosses were isolated as uncompacted 5–7 cell morulae 56 hr p.c. The embryos were maintained in culture for 2–2.5 days and subsequently genotyped by PCR (Fig. (Fig.11d). During this time period, normal morulae first compact (8–16 cell stage) and then differentiate to blastocysts with the formation of an inner cell mass and the outer trophoblast cells (33). Homozygous mutant embryos initially were indistinguishable from wild-type and heterozygous embryos. At approximately 80 hr p.c., however, mutant embryos showed signs of aberrant development. Although morula compaction proceeded in most mutant embryos (Fig. (Fig.22f), the spherical shape of the embryos was subsequently lost and they appeared shrunken. In contrast to control embryos (Fig. (Fig.22 ad), mutant embryos failed to form a blastocoel cavity. At later time points the embryos decompacted (Fig. (Fig.22g) and became progressively more disorganized and fragmented, often with bleb-like protuberances (Fig. (Fig.22h). At 90–100 hr p.c., extensive cellular degeneration occurred in all mutant embryos (Fig. (Fig.22i). TUNEL staining of such embryos revealed a high number of intensely labeled cells indicative of apoptotic cell death (Fig. (Fig.22k). In contrast, heterozygous and wild-type blastocysts only revealed very few apoptotic cells (34) (Fig. (Fig.22e). These experiments confirmed that homozygous SMN disruption in the mouse leads to developmental arrest and death prior to implantation (Table (Table11).

SMN mRNA is highly expressed in oocytes in the ovary of adult mice as shown by lacZ expression (Fig. (Fig.33a). This would indicate that the SMN protein is made in oocytes of mothers with an intact gene copy and may be transferred to and used by early embryonic cells during the first rounds of division. The persistence of maternally produced SMN protein in the embryo could explain the normal development of SMN deleted embryos to the early morula stage despite deletion of the SMN gene in the embryo. Embryonic expression of SMN became appreciable at the late morula stage as assessed by detection of β-galactosidase activity in embryos generated by mating heterozygous males with wild-type females (Fig. (Fig.33 b and c). Thus, the death of mutant embryos may coincide with depletion of maternal SMN in these embryos.

Figure 3
SMN+/− lacZ Expression. (a) Strong expression was found in oocytes of pregnant mare serum gonadotrophin-treated female heterozygotes. (b and c) Embryo lacZ expression. Heterozygous males were mated to wild-type females to determine the ...

Implications of the Murine SMN−/− Phenotype for Human SMA.

These results show that complete loss of SMN in the mouse leads to massive cell death during early embryonic development. The striking difference between the phenotype resulting from loss of the SMN gene in mouse and human could be explained by gene copy number: the mouse genome contains only a single SMN gene copy, whereas the human genome contains two copies at the same locus with nearly identical coding sequence. Both human copies are expressed (10). Loss of the centromeric copy of the gene is not associated with a recognizable phenotype (10, 1719). Genetic data, in particular the identification of healthy individuals with homozygous deletion of the telomeric copy (16, 17, 20, 21), suggest functional redundancy in humans. Although full compensation of SMNtel loss is very rare, the regulation of gene expression of the centromeric SMN could have influence on the disease. This corresponds to the enormous variability in disease severity (1, 5, 8) and suggests that, in the small minority of asymptomatic cases with homozygous SMNtel deletion (16, 17, 20, 21), expression of the centromeric copy may fully compensate for loss of the telomeric gene. Indeed, an association of an increased copy number of the centromeric gene with milder forms of SMA has been observed in a recent study (17). Moreover, homozygous deletion of both genes has not been observed in humans (10, 17, 19). To investigate whether SMN gene dose has an effect on postnatal motoneuron survival in mice, we have quantitated motoneurons in the facial nucleus of 5-month-old SMN+/+ and SMN+/− mice. In SMN+/+ mice, 2,394 ± 65 (mean ± SEM, n = 5) motoneurons were counted; in SMN+/− mice 2,486 ± 253 (mean ± SEM, n = 4) motoneurons were counted. The difference was not statistically significant (P > 0.05, Student’s t test), indicating that a 50% reduction of gene dosage for SMN in mice does not lead to degeneration of motoneurons.

The clinical appearance of human spinal muscular atrophy could be explained by the presence and expression of the centromeric copy in human. Thus, the telomeric SMN gene defect may cause cell death and dysfunction in SMA patients only in such cells where the centromeric copy is not expressed in sufficient quantities, most probably in motor neurons.

A recent study has identified the SMN protein as a constituent of nucleosomal structures (23). Using the yeast two-hybrid system, SMN has been found to associate with the RGG box of heterogeneous nuclear ribonucleoprotein U and fibrillarin. Additional immunohistochemical evidence suggests that it is localized in specific nucleosomes called gems that are associated with coiled bodies (23). The function of these nucleosomal structures is not clear, but it is attractive to speculate that they are involved in the complex mechanism of nuclear RNA processing (23). The massive cell death detectable in SMN-deficient mouse embryos is indeed compatible with such an essential cellular function of the SMN gene product.

Acknowledgments

We thank Timo Grimm, Matthew Digby, and Judith Melki for helpful discussions; Thomas Boehm for the gift of the λPS library; Bettina Holtmann for help with the embryo analysis; Boris Böddinghaus for help with the lacZ stain and providing RNA samples. We also thank Michaela Lederer and Michaela Pfister for technical assistance generating sequence data, and Helga Brünner, Bianca Hagemann, Luise Anderson, and Vanessa McGilliard for mouse husbandry. This work was supported by the Deutsche Forschungsgemeinschaft (Klinische Forschergruppe Neuroregeneration, To 61/8–1) and the Biotechnology and Biological Sciences Research Council (United Kingdom).

Footnotes

This paper was submitted directly (Track II) to the Proceedings office.

Abbreviations: SMN, survival motor neuron; NAIP, neuronal apoptosis inhibitory protein; SMA, spinal muscular atrophy; SMNtel, telomeric SMN copy; SMNcentr, centromeric SMN copy; p.c., post conception; ES, embryonic stem; E, embryonic day.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. Y12835).

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