Human Molecular Genetics, 2001, Vol. 10, No. 23 2727-2736
© 2001 Oxford University Press
Regulation of murine survival motor neuron (Smn) protein levels by modifying Smn exon 7 splicing
1Ottawa Health Research Institute and The University of Ottawa Center for Neuromuscular Disease, Ottawa, Ontario K1H 8L6, Canada, 2Department of Biology, Arizona State University, Tempe, AZ 85287-1501, USA, 3Centre de Recherche, Hôpital Ste-Justine, Montréal, Québec H3T 1C5, Canada, 4Department of Dermatology, New England Medical Center and Tufts University School of Medicine, Boston, MA 02111, USA and 5Department of Cellular and Molecular Medicine, The University of Ottawa, Ottawa, Ontario, Canada
Received August 3, 2001; Revised and Accepted September 9, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Proximal spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron gene (SMN1). In humans, two nearly identical copies of SMN exist and differ only by a single non-polymorphic C
T nucleotide transition in exon 7. SMN1 contains a ‘C’ nucleotide at the +6 position of exon 7 and produces primarily full-length SMN transcripts, whereas SMN2 contains a ‘T’ nucleotide and produces high levels of a transcript that lacks exon 7 and a low level of full-length SMN transcripts. All SMA patients lack a functional SMN1 gene but retain at least one copy of SMN2, suggesting that the low level of full-length protein produced from SMN2 is sufficient for all cell types except motor neurons. The murine Smn gene is not duplicated or alternatively spliced. It resembles SMN1 in that the critical exon 7 +6 ‘C’ nucleotide is conserved. We have generated Smn minigenes containing either wild-type Smn exon 7 or an altered exon 7 containing the CT nucleotide transition to mimic SMN2. When expressed in cultured cells or transgenic mice, the wild-type minigene produced only full-length transcripts whereas the modified minigene alternatively spliced exon 7. Furthermore, Smn exon 7 contains a critical AG-rich exonic splice enhancer sequence (ESE) analogous to the human ESE within SMN exon 7, and subtle mutations within the mESE caused a variation in Smn transcript levels. In summary, we show for the first time that the murine Smn locus can be induced to alternatively splice exon 7. These results demonstrate that SMN protein levels can be varied in the mouse by the introduction of specific mutations at the endogenous Smn locus and thereby lay the foundation for developing animals that closely ‘resemble’ SMA patients. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Proximal spinal muscular atrophy (SMA) is a clinically heterogeneous group of disorders that represent the second most common human autosomal recessive disorder after cystic fibrosis (1). The condition is characterized by the loss of
motor neurons in the spinal cord. Motor neuron degeneration affects voluntary movement and presents as proximal, symmetrical limb and trunk muscle weakness, resulting in respiratory distress and ultimately death. All three major forms of SMA (type I/severe, type II/intermediate and type III/mild) are caused by deletions or mutations of the survival motor neuron (SMN) gene (2). As a result of a recent duplication event on 5q13 (3), humans contain two nearly identical copies of the SMN gene, SMN1 and SMN2; however, only mutations in SMN1 are responsible for SMA (2,4–13). The critical difference between SMN1 and SMN2 is a silent non-polymorphic nucleotide transition (CT) in SMN exon 7 (14,15) that may inhibit one of the splicing regulatory elements present within this exon (16). SMN1 contains a ‘C’ nucleotide at position 6 of exon 7 and produces predominantly full-length transcripts (FLSMN), whereas SMN2 contains a ‘T’ nucleotide at this position. This nucleotide transition in the SMN2 gene results in the production of small amounts of FLSMN (10%) and high levels (
90%) of a differentially spliced form of SMN2 that lacks exon 7 (
7SMN). The resulting protein, SMN7, is less stable and cannot oligomerize or self-associate as well as the protein produced from FLSMN transcripts (16,17), which explains why the SMN2 gene cannot compensate for the homozygous loss of SMN1. However, it is clear from patient studies that SMN2 can modify SMA disease severity in a dose-dependent manner (18–20). In contrast to humans, the mouse Smn gene is not duplicated or alternatively spliced (21,22), and recent studies utilizing mice to assess SMN gene function in vivo, have provided the most compelling evidence for the importance of FLSMN and hence exon 7 (23–25). These studies have also demonstrated that SMN2 is capable of rescuing the embryonic lethality in Smn–/– mice; however, a high copy number of the SMN2 gene is required for complete rescue (24,25). Given these findings, modulation of SMN2 expression might present a possible therapeutic approach towards attenuating the SMA phenotype.
Our goal is to create an allelic series of Smn mice at the endogenous murine Smn locus, which would mimic human SMN2 by producing both FLSmn and 7Smn transcripts and protein. These mice would resemble the situation in SMA patients and could be used to systematically titrate SMN protein levels and determine the minimal amount required for normal postnatal development and maintenance of motor neurons. To determine if we could develop this type of animal, we have generated Smn minigenes containing either wild-type Smn exon 7 or an altered exon 7 containing the CT nucleotide transition found in the SMN2 gene. When expressed in cultured cells or transgenic mice, the wild-type minigene produces only full-length transcripts whereas the modified minigene alternatively spliced at exon 7. We have also analyzed the regulation of Smn exon 7 and show that it contains an exonic splice enhancer sequence (ESE) that is structurally and functionally similar to the human AG-rich splice enhancer within SMN exon 7. By creating subtle mutations within the ESE, Smn exon 7 transcript levels were varied in the minigene and in transgenic animals that harbored similar minigene constructs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Analysis of murine Smn minigenes in a cell culture system
We have previously cloned and sequenced the entire Smn gene (26). Based upon this, plasmid-based minigenes were constructed to determine whether the murine Smn locus can be induced to alternatively splice Smn exon 7. To this end, a 5.7 kb SalI genomic fragment was cloned into the mammalian expression vector pCI, downstream of the cytomegalovirus (CMV) promoter to create the minigene pSmnWt (Fig. 1B). This construct is analogous to the human SMN1 gene. Exon 7 is highly conserved at the nucleotide level and the endogenous murine Smn gene and human SMN1 contain a ‘C’ at the +6 position within exon 7. To generate a murine minigene that was similar to human SMN2, site-directed mutagenesis was used to introduce the C
T nucleotide transition at position +6 within Smn exon 7. The resulting minigene was named pSmn(C-T) (Fig. 1B). As controls, we used the empty vector and the human minigenes pSMN1 and pSMN2 (14) (Fig. 1A). Plasmids were transfected into C2C12 cells, a murine skeletal muscle cell line, and RNA was extracted 48 h post-transfection. RT–PCR analysis was performed using oligonucleotide primers specific to transcript sequences within the plasmid backbone, which ensured that only plasmid-based transcripts would be amplified (Fig. 1D, lane 4). As expected, pSMN1 produced abundant FLSMN transcript with no detectable 7SMN (Fig. 1C, lane 2 and E), and pSMN2 produced a low level of FLSMN and abundant 7SMN transcript (Fig. 1C, lane 3 and E). Analysis of the murine minigene constructs indicated that the pSmnWt minigene recapitulated the endogenous Smn splicing pattern: only FLSmn was produced (Fig. 1D, lane 7). In contrast, the pSmn(C-T) minigene produced both FLSmn and transcripts lacking exon 7 (Fig. 1D, lane 6). Subcloning and sequencing confirmed the identity of the differentially spliced product. Interestingly, this analysis revealed that the mouse 7Smn transcripts used the normal 5' splice donor site of exon 6 but used a cryptic 3' splice acceptor site in exon 8 that is 45 nucleotides distal to the acceptor typically used when exon 7 is present (Fig. 1F). However, this does not change the C-terminus of the encoded protein, SmnC15, since the first 45 nucleotides of exon 8 are repeated in tandem. Thus, the introduction of a single CT nucleotide transition in an otherwise wild-type background is sufficient to create a murine locus that mimics the human SMN2 locus and is able to produce both FLSmn and 7Smn transcripts.
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Using a semi-quantitative RT–PCR approach that we have previously described (16), the percentage of FLSmn and
7Smn from at least seven independent transient transfection experiments was determined to be 67.3 and 32.7% ± 2.8, respectively (Table 1). For ease of reporting only the percentage of FLSmn will be shown. The amount of DNA used for transfection had no effect upon the transcripts produced or the relative amount of FLSmn and 7Smn transcripts (Fig. 2A, lanes 5–10 and data not shown). Additionally, RNA processing of the murine minigene transcripts was assessed in several different cell lines, including Cos-1, HeLa, H9, A9 and U20S (monkey kidney, human kidney, rat cardiomyocyte, murine fibroblasts and human osteosarcoma, respectively). In all instances, the level of FLSmn and 7Smn transcripts was similar (Fig. 2B). Therefore, these results demonstrate that the amount of alternatively spliced product produced from pSmn(C-T) is not cell-type or species specific. The human SMN2 splicing pattern has previously been reported to be unaffected by similar contextual changes (14).
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The level of FLSMN transcripts produced from the minigenes pSMN2 (human) and pSmn(C-T) (mouse) in transient transfections is not equivalent. FLSMN transcripts account for
15–20% of SMN2-derived transcripts (2,16,27), which is consistent with our study (Table 1). To confirm the expression pattern of the murine minigene, we have created pooled stable cell lines as well as clonal isolates for pSMN2 and pSmn(C-T) in both C2C12 and Cos-1 cells. We observed no difference in the level of FLSMN from the pSMN2 minigene in stable cell lines versus transiently transfected cells. However, the level of FLSmn in pSmn(C-T) stable cell lines was different than that obtained in transient transfection assays. We observed a relative decrease in the abundance of FLSmn and a corresponding increase in 7Smn in stably transfected cells (46.0% FLSmn ± 5.0) compared to transiently transfected cells (67.3% FLSmn ± 2.8).
Analysis of Smn minigenes in transgenic mice
To determine whether the Smn minigenes can recapitulate in vivo what was observed in cell culture, we generated transgenic mice by microinjecting pSmnWt and pSmn(C-T) minigenes into the pronuclei of fertilized mouse eggs. In initial experiments, potential transgenic founders were killed at embryonic day 14.5 (E14.5). The yolk sac from each embryo was used to extract DNA for genotyping and the embryo itself was separated into head, liver and body for RNA analysis. Transgenic embryos were identified by PCR using a sense oligonucleotide in intron 7 and an antisense oligonucleotide in the vector backbone. In this way, only those animals harboring the minigene construct would be positive by PCR. All transgenic founder embryos derived from the pSmnWt minigene expressed only FLSmn transcripts (Fig. 3; Table 2). In contrast, pSmn(C-T) transgenic embryos expressed FLSmn and 7Smn transcripts (Fig. 3) at levels similar to those achieved in transient transfection assays (Table 2). An additional transcript that lacked exons 6 and 7 (6,7Smn) was observed in one cDNA sample (Fig. 3, lane 8). This aberrant transcript used the normal 5' and 3' splice sites of exons 5 and 8, respectively (data not shown).
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Next, live-born transgenic founder mice were generated with the pSmn(C-T) minigene construct to determine if the minigene-derived transcripts or the relative ratio of full-length/exon-skipped products were affected temporally or spatially. Four transgenic animals (508, 509, 531 and 543) were identified by PCR and bred to establish independent lines. Each line was assessed for transgene expression pre- and postnatally using a semi-quantitative RT–PCR assay (16). One line (509) did not express the transgene during embryonic stages or postnatally (data not shown). The remaining three lines expressed the transgene at high (543), medium (508) or low (531) levels (Fig. 4). Transgene expression from line 531 was only detected when radioactivity was incorporated into the RT–PCR reaction and 40 cycles of amplification were performed. Therefore, this line was not characterized further. Lines 508 and 543 expressed varying levels of the transgene depending upon the tissue, although expression was detected in all tissues analyzed (Fig. 4). Interestingly, in those tissues where the transgene was expressed at very low levels, the relative ratio of FLSmn:
7Smn was nearly equivalent. Similar results were obtained from either single or pooled cell clones of pSmn(C-T) made in Cos-1 and C2C12 cells (data not shown).
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Line 543, the highest expressing line, was analyzed in greater detail using animals that were heterozygous for the transgene. The cDNA from head, liver and body of E14.5 transgenic embryos were compared to five tissues, kidney, heart, spinal cord, skeletal muscle and brain, collected from transgenic heterozygotes at postnatal days P15, P30 and P>180 (data not shown). We did not observe any difference in the percentage of FLSmn or in the transcripts that were produced, indicating that for at least this line, there was no temporal and spatial influence on transgene expression.
Smn contains an exonic splice enhancer that is essential for the generation of full-length transcripts
Typically, the sequences at the 3' and 5' exon–intron boundaries determine the accurate recognition and splicing of exons. However, additional cis regulatory elements can be present. These include ESEs, which serve as binding sites for a class of regulatory proteins known as SR proteins. ESEs can facilitate the inclusion of the exon they reside in and can be found in regulated or constitutively expressed exons (28–30). We have previously shown that several regulatory splicing elements exist within human SMN exon 7 (16). The most critical is an AG-rich enhancer (hSE2) within the central region of this exon that is required for the constitutive inclusion of exon 7 in vivo and in vitro (16). Comparative sequence analysis of human and mouse SMN exon 7 indicates that the exon and flanking intronic sequence are highly conserved (Fig. 5A). Based upon sequence comparison, we speculated that similar splicing regulatory elements exist within exon 7 of the murine Smn gene and the most important of these would be the AG-rich enhancer (mSE2) element within the central region of the exon.
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To determine whether the AG-rich region of Smn exon 7 is required for the production of full-length transcripts, a series of mutated Smn minigenes was analyzed by RT–PCR following transient expression. The murine SE2 element (mSE2) was divided into two subdomains: mSE2A and mSE2B (Fig. 5A). Since ESEs are particularly sensitive to U residue substitutions (31–33), derivative minigene constructs were created from pSmnWt by introducing three consecutive T nucleotides (U residue in the RNA sequence) into each subdomain. The resulting minigenes were assessed for their effect upon exon 7 splicing following transient expression in U20S cells. The relative ratio of FLSmn:
7Smn for each mSE mutant is summarized in Table 1. Mutation of mSE2A had a modest effect upon Smn exon 7 splicing and produced even less 7 transcripts than pSmn(C-T) (Fig. 5B, compare lanes 1 and 3). In contrast, mutation of mSE2B resulted in 70% of Smn minigene transcripts lacking exon 7 (Fig. 5B, lane 4). Furthermore, a small in-frame deletion of the entire mSE2 element resulted in the production of high levels 7Smn and undetectable levels of the full-length transcript (Fig. 5B, lane 5). To confirm the role of the AG-rich enhancer region, transgenic mice were generated that harbored the various mutated minigenes. Similar levels of FLSmn and 7Smn were detected from transgenic mice carrying the minigenes, confirming the results of the transient expression experiments (Fig. 5C; Table 2). To demonstrate that mSE2 is a bona fide ESE, we constructed chimeric in vitro splicing templates with the putative ESE from Smn exon 7 and the previously described Drosophila melanogaster double-sex cassette (28,31,33). In vitro splicing from exon 3 to 4 only occurs in the presence of strong downstream splice enhancers. When a single copy of the mSE2 region is fused downstream of the dsx sequences, a low level of splicing was stimulated compared to a single copy of the analogous human region. However, insertion of three or five copies of either the murine or human ESE elements stimulated high levels of splicing, confirming that mSE2 is a true splice enhancer (Fig. 6).
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Taken together, the above results demonstrate that the AG-rich region of murine Smn exon 7, like its human counterpart, functions as an ESE and is required for the constitutive inclusion of exon 7.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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To properly assess potential therapeutic agents for their efficacy and safety in the treatment of SMA, or any disease for that matter, relevant animal models need to be available. In the case of SMA, model animals should produce both FLSmn and
7Smn transcripts and have a motor neuron deficit. Ideally, one would like a panel of animals that vary in phenotype from severe to mild.
The present study was designed to determine if the endogenous murine Smn locus can be induced to alternatively splice exon 7 and produce FLSmn and 7Smn. We also sought to determine if the regulation of Smn exon 7 splicing is conserved since one possible therapeutic approach to SMA would be to suppress alternative splicing of SMN2 exon 7. This was accomplished using a minigene system where we modified the wild-type Smn locus to resemble human SMN2. This type of model system has been used previously to dissect the elements that regulate the alternative splicing of a number of different genes, including human SMN (14,34–36).
The murine Smn locus resembles human SMN1 in that it contains a ‘C’ nucleotide at the +6 position of exon 7 and is not alternatively spliced (21,22). We have created a wild-type murine minigene, pSmnWt, and have shown in transient transfection assays, that it recapitulates the splicing pattern observed for the endogenous murine Smn locus and is not alternatively spliced. A murine minigene that mimics human SMN2, pSmn(C-T), was generated by creating the CT nucleotide transition in exon 7 in an otherwise wild-type background. Transient expression of this construct produced both FLSmn and 7Smn. Thus, we show for the first time that the murine Smn locus can be induced to alternatively splice Smn exon 7 and produce both FLSmn and 7Smn transcripts.
Analysis of the 7Smn transcript revealed the use of a cryptic splice site 45 nucleotides distal to the normal 5' splice acceptor site of exon 8. Interestingly, the first 45 nucleotides of murine Smn exon 8 are repeated in tandem. Consequently, the protein, SmnC15, is the same whether the normal or cryptic exon 8 splice acceptor site is used. Frugier et al. (23) also show identical differential splicing of exon 7 at the endogenous Smn locus. Therefore, the cryptic splice site selection of 7Smn transcripts is not an artifact of our minigene system and supports the use of the pSmn(C-T) plasmid to study the regulation of Smn exon 7 splicing.
Both human and murine ‘SMN2’ minigenes produced FLSMN and 7SMN transcripts. However, the level of FLSMN transcripts produced from pSMN2, the human SMN2 minigene, and pSmn(C-T), the murine minigene created to mimic human SMN2, were not similar. Several reports, including this study, have shown that the amount of FLSMN produced from pSMN2 is 15–20% (14,15,27), which is similar to the endogenous SMN2 locus. Given the high degree of sequence similarity between human and mouse SMN exon 7, we expected that the level of FLSmn produced from pSmn(C-T) would be similar to its human counterpart; however, we observed that 70% of the transcripts produced from the minigene were FLSmn. The observed difference in exon 7 skipping may be due to the fact that the murine minigenes only have a partial exon 8 sequence, whereas the human minigenes contain a complete exon 8. This seems less likely since the majority of SMN exon 8 is dispensible for normal SMN1 and SMN2 splicing regulation (C.L.Lorson, unpublished data). Alternatively, although exon 7 is conserved between human and mouse, the flanking intron size and sequence is not. This may also contribute to the observed quantitative difference in human and murine SMN exon 7 skipping. A relationship between the SMN exon 7 poly-pyrimidine tract and the AG-rich ESE (SE2) has previously been identified, further suggesting that the differences in the upstream sequences may act to regulate splicing. Taken together, these results demonstrate that the murine Smn locus can be induced to alternatively splice exon 7 simply by exchanging the ‘C’ nucleotide at the +6 position of exon 7 for a ‘T’ nucleotide.
Sequence comparison between human and mouse SMN exon 7 indicate that they are highly conserved and that the SE2 element, which is critical for exon inclusion, is identical. Mutation analysis of the mSE2 element showed that it is required for the constitutive inclusion of Smn exon 7 and that in its natural context it is an ESE sequence. A hallmark of bona fide splice enhancers is that they can function in a heterologous context. To determine whether the mSE2 element was a true splice enhancer, chimeric in vitro splicing templates were constructed with the putative ESE from Smn exon 7 and the previously described D.melanogaster double-sex in vitro splicing cassette (28,31,33). In vitro splicing from exon 3 to 4 only occurs in the presence of strong downstream splice enhancers. The in vitro splicing results confirm that the mSE2 region acts as an ESE. In addition, its relative strength is similar when compared with the human SE2 element. This is important since a number of therapeutic approaches involve the stimulation of FLSMN expression from SMN2, and SE2 modifying factors are likely therapeutic targets.
This work indicates that murine Smn can be induced to skip exon 7 and that FLSmn expression can be varied from 100 to 0% by making subtle mutations in an otherwise wild-type background. To confirm the relative ratio of FLSmn:7Smn transcripts that we observed in cell culture experiments, we assessed all of our minigenes in vivo using transgenic mice. These results were consistent with the results from our cell culture experiments, although minor differences in the percentage of FLSmn were observed. We also generated transgenic lines using the minigene pSmn(C-T). Analysis of the highest expressing line, tg543, indicated that there was no difference temporally or spatially in the percentage of FLSmn or in the transcripts that were produced. This suggests that the factors that regulate Smn exon 7 processing are ubiquitously expressed and that the decreased level of SMN expression observed postnatally (18,37–39) is not due to alternative splicing of Smn exon 7 but most likely occurs through regulating the SMN promoter.
The work presented here has important implications for developing Smn alleles that survive postnatally and mimic human SMN2 by producing both FLSmn and 7Smn. Our results suggest that the most interesting alleles to generate would be the CT nucleotide transition and the mSE2B mutation. The generation of animals with these subtle mutations, as well as compound heterozygotes with the null Smn–/– allele, would allow the titration of Smn protein levels and the systematic determination of the effect of SMN dosage on development.
Overall, we show for the first time that the murine Smn locus can be induced to alternatively splice exon 7 and that Smn contains similar regulatory elements compared to human SMN exon 7. Our results lay the foundation for using a genetic approach to systematically determine the minimal amount of SMN protein required for normal development and provide a relevant animal model for SMA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmids
The human minigene plasmids pSMN1 and pSMN2 were described previously (14). To construct the murine wild-type minigene, pSmnWt, a 5.7 kb SalI genomic fragment from BAC 20g19 (21) was cloned into the SalI site of the plasmid, pCI (Promega), which had been modified to remove the NheI–XbaI restriction sites. The SalI genomic fragment contains a small portion of the 3' end of Smn intron 4, Smn exons 5–8 (91 bp) and their corresponding intronic regions.
To facilitate the construction of plasmids pSmn(C-T), mSE2A, mSE2B and mSE2 a KpnI–NotI fragment from pSmnWt, containing Smn exons 7 and 8, was subcloned into the KpnI–NotI sites of pBSPT KS+. The resulting plasmid was used as a template to perform site-directed mutagenesis using either the Quickchange kit (Stratagene) [pSmn(C-T)] or the two-step PCR overlap extension method (mSE2A, mSE2B, mSE2). The resulting plasmids were cloned into the KpnI–NotI sites of pSmnWt. The resultant plasmids were sequenced to ensure that no extraneous mutations existed which might alter the splicing of the minigenes. The following primer sets were used to generate the indicated mutations (underlined): pSmn(C-T), 5'-CTT TAC AGG GTT TTA GAC AAA AT-3', 5'-ATT TTG TCT AAA ACC CTG TAA AG-3'; mSE2A, 5'-GAC AAA ATA AAA AAT TTG GAA AGT GCT CAC ATA C-3', 5'-GTA TGT GAG CAC TTT CCA AAT TTT TTA T TT TGT C-3'; mSE2B, 5'-GAC AAA ATA AAA AAG AAT TTA AGT GCT CAC ATA C-3', 5'-GTA TGT GAG CAC TTA AAT TCT TTT TTA TTT TGT C-3'; mSE2, 5'-GGT TTC AGA CAA AAT AAA AAA TGC TCA CAT ACA AAT TAA G-3', 5'-CTT AAT TTG TAT GTG AGC TTT TTT TAT TTT GTC TGA AAC C-3'.
In vitro splicing cassettes were constructed by annealing the following pairs of complimentary oligonucleotides that created XbaI and HindIII overhanging ends between the XbaI and HindIII sites of 3014, a plasmid that contains the previously described Drosophila double-sex splicing cassette (33). The XbaI and HindIII overhangs are underlined: mSEx1, 5'-CTAGAAAGAAGGAAAGA-3', 5'-AGCTTCTTTCCTTCTTT-3'; mSEx3, CTAGAAAGAAGGAAAGAAGAAGGAAAGAAGAAGGAAAGA-3', 5'-AGCTT CTTTCCTTCTTCTTTCCTTCTTCTTTCCTTCTTT-3'; mSEx5, 5'-CTAGAAAGAAGGAAAGAAGAAGGAAAGAAGAAGGAAAGAAGAAGGAA AGAAGAAGGAAAGA-3', 5'-AGCTTCTTTCCTTCTTCTTTCCTTCTTCTTTCCTTCTTCTTTCCTTCTTCTTTCCTTCTTT-3'.
Cell culture and transfections
Cos-1, C2C12, U20S, H9, A9 and HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose that had been supplemented with 10% fetal bovine serum (FBS). Twenty-four hours prior to transfection, cells were seeded at a density of 1.0 x 105 cells/well in 6-well culture plates. All minigene constructs were transfected into the above cell types using Superfect Reagent (Qiagen) and 0.05–2.0 µg of DNA according to the manufacturer’s instructions. Briefly, the appropriate amount of DNA was diluted in 100 µl of plain DMEM and then 8 µl of Superfect was added, tapped, mixed and incubated for 30 min. Complete media (600 µl) was added to each tube and the final mix was added drop wise to each well of the culture plate that had just been rinsed with 1x PBS. Four hours post-transfection the media volume was increased to 2 ml/well and the cells were grown for an additional 48 h.
RNA isolation and RT–PCR
Tissues and transfected cells were lysed in buffer RLT (Qiagen) and total cellular RNA was purified using the RNeasy Mini Kit (Qiagen). First-strand cDNA was synthesized in a 20 µl reaction volume using oligo(dT) and MMLV reverse transcriptase (Life Technologies) according to the manufacturer’s instructions. PCR amplification analysis of plasmid-derived cDNAs was performed using vector-specific primers pCI forward (5'-GCT AAC GCA GTC AGT GCT TC-3') and pCI reverse (5'-GTA TCT TAT CAT GTC TGC TCG-3'). For each amplification, an aliquot (1–5 µl) of first-strand cDNA was used. PCR was performed in a total volume of 25 µl that contained 20 mM Tris–HCl pH 8.4, 50 mM KCl, 50 mM MgCl2, 4 µM each primer, 200 µM dNTPs and 1 U Taq DNA polymerase (Life Technologies). Amplification conditions were as follows: an initial 3 min denaturation step at 94°C, followed by 29 cycles of 94°C for 30 s, 56°C for 45 s and 72°C for 45 s in an Eppendorf Mastercycler. The resulting products were electrophoresed in a 2% agarose/1x TBE gel containing ethidium bromide. Semi-quantitative RT–PCR analysis of plasmid-derived transcripts was performed as described previously (16) except that 18 and 25 cycles of amplification were used for cDNA isolated from tissue-cultured cells and animal tissues, respectively. Quantitations were performed on an Instantimager (Packard Biosciences); FL and 7 transcripts were quantitated and expressed as a percentage relative to full-length expression within the same reaction ± SD.
Production of transgenic embryos and mice
The murine Smn minigenes described here [pSmnWt, pSmn(C-T), mSE2A, mSE2B and mSE2] were linearized by PvuI digestion, which removed 2 kb of extraneous vector sequence. The digested DNAs were electrophoresed through 0.8%/1x TBE agarose gels and 9 kb fragments were excised and purified using the Qiaex II gel extraction kit (Qiagen). The resulting DNA was diluted to a final concentration of 3 ng/µl in filtered TE buffer (10 mM Tris–HCl pH 7.4, 0.1 mM EDTA) and injected into the pronuclei of C57Bl6/C3H fertilized eggs. Surviving embryos were implanted into pseudopregnant females using standard methods (40). Transgenic animals were either studied as founders isolated 14.5 days (E14.5) after microinjection or stable lines were established and F1 animals were analyzed. The genotype of animals was determined by PCR analysis using DNA isolated from yolk sacs or tail DNA and primers 277.2 (5'-GGC GCT GTC CTG GAA CC-3') and pCI Rev (described above). The reaction and cycling conditions were the same as those described above. Amplifications products were resolved on 2%/1x TBE agarose gels and scored for the presence or absence of an 400 bp product that is present only in transgenic animals.
Cloning and sequencing of RT–PCR products
The PCR-amplified fragments derived from the plasmid-based transcripts were cloned using the TA cloning vector, pCR2.1 (Invitrogen). To verify the amplified products, at least three subclones from each were sequenced using an ABI 377 sequencer.
In vitro splicing
The Promega RNA Splicing System was used as recommended by the manufacturer. Briefly, in vitro splicing reactions were performed at 30°C for 3 h using 10 µl of HeLa nuclear extract and 10 ng of gel-purified [32P]dUTP-labeled RNA templates. Reaction products were phenol extracted twice, precipitated and resolved on a 6% sequencing gel. Positive control reactions resulted in similarly sized splice products and DNA sequencing reactions were used to identify the final products.
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ACKNOWLEDGEMENTS |
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We would like to thank Drs V.Wallace and D.Bulman for critically reading this manuscript and Carl Baker for the in vitro splicing construct. Funding for these studies was provided by grants from the Families of Spinal Muscular Atrophy (FSMA), the Canadian Institutes of Health Research (CIHR, grant no. MOP-38040), the Muscular Dystrophy Association (C.L.L. and E.J.A.) and the National Institutes of Health (C.L.L., RO1 NS41584-01; E.J.A., RO1 NS40275). C.J.D. is supported by postdoctoral fellowships from FSMA and the Muscular Dystrophy Association (USA).
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada. Tel: +1 613 737 8707; Fax: +1 613 737 8803; Email: rkothary@ohri.ca
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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