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. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: Dev Dyn. 2008 Aug;237(8):2268–2278. doi: 10.1002/dvdy.21642

The identification and characterization of the porcine (Sus scrofa) Survival Motor Neuron (SMN1) gene

An animal model for therapeutic studies

Monique A Lorson a,*, Lee D Spate b, Randall S Prather b, Christian L Lorson a
PMCID: PMC2556073  NIHMSID: NIHMS65001  PMID: 18651653

Abstract

Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder that is characterized by the degeneration of the motor neurons of the spinal cord leading to muscle atrophy. SMA is a result of a loss-of-function of the gene SMN1. We have chosen to generate a transgenic swine model of SMA for the development and testing of therapeutics and evaluation of toxicology. To this end, we report the first cloning and identification of the swine SMN1 gene and show that there is significant sequence homology between swine and human SMN throughout the coding region. RT-PCR results demonstrated slight changes in SMN RNA expression during development and in different tissues. In contrast, protein expression profiles were dramatically different based upon different tissues and developmental stages, consistent with human SMN expression. Porcine SMN localization is consistent with human SMN, localizing diffusely within the cytoplasm and in punctate nuclear structures characteristic of nuclear gems. Importantly, transient transfection of porcine SMN1 in 3813 SMA type 1 fibroblasts demonstrate that porcine SMN1 can rescue the deficiency of SMN protein and gem formation in these cells. These studies provide the first characterization of the porcine SMN1 gene and SMN protein and suggest that a transgenic swine SMA model is feasible.

Keywords: SMN1 expression, Spinal Muscular Atrophy, swine, animal model

INTRODUCTION

Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder that is characterized by the degeneration of the anterior horn cells of the spinal cord leading to muscle atrophy. SMA is the second most common autosomal recessive disorder after Cystic Fibrosis and is the most common inherited motor neuron disease. SMA occurs in approximately 1:8,000 live births and has a carrier frequency of ∼1:45. There are three SMA clinical groups based on age of clinical onset and disease severity. Patients with SMA type I (severe) show disease onset around birth, never sit or walk and usually die of respiratory distress within two years of age. SMA type II patients (intermediate) typically show disease onset after six months. Patients can sit but never walk without assistance and have a shortened lifespan. SMA type III patients (least severe) typically show disease onset after eighteen months and can stand and walk but later require ambulatory assistance in adolescence (Wirth et al., 1995b; Crawford and Pardo, 1996; Wirth et al., 1999; Wirth, 2000).

The gene responsible for SMA is called survival motor neuron-1 (SMN1). SMN1 alleles predominantly produce full-length SMN transcripts of nine exons. The translation termination codon is at the 3′ end of exon 7 and exon 8 is non-coding. A human-specific copy gene is present on the same region of chromosome 5q called SMN2 which is a result of a large duplication and inversion event (Lefebvre et al., 1995; Roy et al., 1995; Wirth et al., 1995a; Rochette et al., 2001). SMN2 is nearly identical to SMN1; however, mutations in SMN2 have no clinical consequence if SMN1 is retained. Low copy number of SMN2 cannot prevent disease development in the absence of SMN1 because the majority of SMN2-derived transcripts are alternatively spliced, resulting in a truncated protein that lacks the 16 amino acids derived from SMN exon 7 (Gennarelli et al., 1995; Lefebvre et al., 1995). A single C to T non-polymorphic nucleotide difference between SMN1 and SMN2 is responsible for the alternative splicing of the SMN transcripts; however, this creates a silent nucleotide transition that does not alter the protein coding capacity of SMN2 (Lorson et al., 1999; Monani et al., 1999; Cartegni and Krainer, 2002). As a result, SMN1 produces almost exclusively full-length SMN while SMN2 produces an alternatively processed product lacking exon 7 (90%) and 10% full-length SMN. Numerous studies have shown that the SMN2-derived protein product (called SMNΔ7) is unstable and nearly non-functional, further demonstrating the critical nature of the SMN exon 7 pre-mRNA splicing (Frugier et al., 2000; Lorson and Androphy, 2000).

The human SMN protein is an ubiquitously expressed protein of 294 amino acids that migrates with a molecular weight of approximately 38 kDa. The SMN protein is found in many cells within the central and peripheral nervous system and is abundant in normal motor neurons (Lefebvre et al., 1995; Battaglia et al., 1997; Novelli et al., 1997; Tizzano et al., 1998; Young et al., 2000a). SMN protein is significantly reduced in SMA affected tissue and primary cell cultures. Why a ubiquitously expressed protein specifically results in a motor neuron deficiency is currently unknown. However, several functions of the SMN protein have been described. The most completely characterized roles for SMN are in snRNP biogenesis. In this function, SMN is found in a “core” complex with several other proteins called gemins (Gemin 2-8) and unrip (Fischer et al., 1997; Liu et al., 1997; Charroux et al., 1999; Charroux et al., 2000; Baccon et al., 2002; Gubitz et al., 2002; Meister et al., 2002; Pellizzoni et al., 2002; Gubitz et al., 2004; Feng et al., 2005; Ma et al., 2005; Shpargel and Matera, 2005; Carissimi et al., 2006). A distinguishing feature of the SMN protein is that it localizes to punctate nuclear bodies originally identified as ‘gems’ (Liu and Dreyfuss, 1996; Young et al., 2000a; Navascues et al., 2004). The number of gems in SMA patient-derived fibroblasts and tissues is linked to disease severity (Coovert et al., 1997). In most cell lines and tissues, the structures originally identified as gems are coincident with Cajal bodies; structures that are highly enriched for RNA processing components including snRNPs and the Cajal body-marker protein p80 coilin (Matera and Frey, 1998; Hebert et al., 2001). SMN is also proposed to function in the transportation of β-actin mRNA in neurites raising the possibility that SMN is involved in the axonal localization and transport of specific mRNA species (Rossoll et al., 2003).

The human genome is unique containing SMN1 and SMN2 genes; however genes analogous to SMN1 are present in all mammals examined to date and in organisms including Caenorhabditis elegans, Danio rerio, Drosophila, and Sacharomyces. The murine homologue is eighty-two percent identical to the human SMN protein and a Smn knock-out results in pre-implantation lethality, suggesting that while SMN performs a critical neuronal-specific function, it is also required for general cellular activities, likely snRNP assembly (Bergin et al., 1997; DiDonato et al., 1997; Schrank et al., 1997). A series of mice have been produced that lack the endogenous Smn (Smn-/-) and contain 2, 4, or 8 copies of the human SMN2 gene. These results and the identification of an unaffected individual with five SMN2 copies and no SMN1 genes, clearly demonstrate that SMA severity decreases as the number of SMN2 copies increases (Monani et al., 2000).

Murine SMA models have proven very valuable to our understanding of SMA. However, these models inherently have limitations, most importantly the short window of disease progression. Therefore, we have chosen to generate a transgenic swine model of SMA specifically for evaluating the efficacy of therapeutics and studying toxicology. The domestic swine Sus scrofa is an important and powerful tool in biomedical research. Swine serve as excellent models for cardiovascular disease, cutaneous pharmacology and toxicology research, and xenotransplantation (Prather et al., 2003; Kolber-Simonds et al., 2004; Vodicka et al., 2005; Lai et al., 2006). Swine are well-suited transgenic models for a number of critical reasons. From a physical standpoint swine body size is similar to humans, cardiovascular metabolism is more similar to humans as compared to mice, the porcine genome is more similar to human than is the mouse, the evolutionary relationship between swine and human is closer than mouse to human despite divergence at a similar time and swine have more similar lifespan to humans than do mice (Wernersson et al., 2005). Presented in this manuscript is the identification of the Sus scrofa SMN1 gene and its expression patterns as a means to validate the appropriateness of producing a swine model for SMA.

RESULTS

Identification of porcine SMN1

As the SMN1 gene has been identified in eukaryotic organisms ranging from yeast to humans, it was predicted that the swine would contain a SMN1 gene. The human SMN1 cDNA sequence was searched against the TIGR (The Institute for Genomic Research) Porcine (Sus scrofa) Gene Index and TC239547 was identified as the putative porcine SMN1 sequence from a set of 29 ESTs. In addition, a SMN1 cDNA was amplified from PK15 cells (porcine kidney) and fetal-derived fibroblast cells by using RT-PCR and sequenced (supplementary data). The SMN1 cDNAs differ slightly in sequence; at positions 309, 394, 842 and 864 there are guanine to adenine substitutions and at position 669 there is an “ACC” deletion which results in the in-frame loss of a single proline from the predicted SMN protein (supplementary data). SMN1 cDNAs from other porcine cells were amplified with these same changes (data not shown). In agreement with these findings, EST sequences reported in the Gene Index also contain these alterations. It is predicted that the differences between TC239547 and the SMN1 cDNA isolated in these studies are polymorphisms. For these studies the PK15 derived cDNA will be referred to as SMN1a and TC239547 will be referred to as SMN1b.

The porcine SMN1 locus was identified using genomic DNA isolated from PK15 and fetal-derived fibroblast cells. Using primers for each SMN1 exon junction, genomic DNA was amplified, cloned and sequenced. To generate overlapping clones, genomic DNA was amplified and sequenced to confirm exon sequence and exon/intron boundaries. Using multiple methods, we are currently unable to amplify intron 1. Comparison of SMN1 genomic sequence from horse, dog, cow and humans demonstrates that intron 1 exceeds 12kb in each of these species. These sequence comparisons suggest that porcine SMN1 intron 1 likely exceeds 12 kb and explains the difficulty in amplifying this sequence. As the genome of Sus scrofa is currently being sequenced, we expect the sequence to be completed in a timely manner. The genomic organization of the porcine SMN1 gene was determined based on the completed sequence and predicted size of intron 1 (Figure 1A). The SMN1 locus spans approximately 27 kb, similar to the size of the human SMN1 locus of approximately 28 kb. Based on sequence identity it is predicted that porcine SMN1 consists of eight coding exons. In keeping with the nomenclature of human SMN1, the exons have been referenced as exons 1, 2a, 2b, 3, 4, 5, 6 and 7. The non-coding exon 8 in human SMN1 bears little sequence identity to porcine SMN1. The introns range in size from 143 bp to ∼12 kb. The porcine SMN1 genomic sequence conforms to the typical GT/AG splice donor and splice acceptor sites.

Fig. 1.

Fig. 1

Fig. 1

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The identification and comparison of porcine SMN1 gene products. A: The organization of the porcine SMN1 locus. Coding exons are indicated as blue boxes and introns are indicated as yellow arrows. The introns range in size from 143 bp to the predicted 12 kb of intron 1. The broken arrow in intron 1 indicates the size is predicted and not to scale. The relative scale of the introns is indicated. B: The alignment of the predicted proteins for mouse, porcine and human SMN. Porcine SMN is 90% identical to human SMN while mouse SMN is 82% identical to human SMN. Amino acids that differ between human and swine are boxed. Exon 2 and Exon 6 self-association domains are indicated with a black line. The Tudor Domain consists of amino acids 91-122. Some SMA patient mutations are indicated with asterisks. The alignments were carried out by using DNASTAR (Lasergene). Graphics were designed using Vector NTI (Invitrogen).

The SMN1a and SMN1b ORFs (open-reading-frame) are 882 and 885 nts respectively and are predicted to encode a protein of 293 (294) amino acids, as does human SMN. The alignment of the mouse, porcine and human SMN proteins is shown in Figure 1B. There is a high degree of identity between porcine and human SMN, 90% identity and 93% similarity, significantly higher than the predicted SMN protein in mouse which shares 82% identity to humans (Figure 1B). The previously identified Tudor Domain and self-association domains are present and highly conserved. The Tudor Domain within human SMN preferentially binds RG-rich proteins including the Sm and Sm-like proteins required for SMN-dependent cytoplasmic core-UsnRNP assembly (Brahms et al., 2001; Boisvert et al., 2002). Self-association of human SMN, mediated through exons 2 and 6 is required for nearly all biochemical functions ascribed for SMN (Lorson et al., 1998; Pellizzoni et al., 1999; Young et al., 2000b).

Expression of the porcine SMN protein

In order to examine SMN expression in porcine cells, western blot analysis was performed on extracts from porcine cell lines PK15 (porcine kidney) and PFT-74 (porcine fallopian tube) and porcine fetal-derived fibroblast cells. Human HeLa cells were used as a control. Membranes were reacted with an anti-SMN monoclonal antibody (Figure 2). The anti-SMN antibodies recognized a protein of the predicted size in the porcine cell lines, fetal-derived fibroblast cells and in the control cells (Figure 2). Actin was used as a loading control. These results suggest that SMN is highly expressed in porcine cells and migrates at approximately 38 kDa, comparable to the human SMN protein.

Fig. 2.

Fig. 2

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Expression of porcine SMN. SMN is abundantly expressed in porcine cells. Expression of SMN was analyzed in three cell types PK15 (porcine kidney), PFT-74 (porcine fallopian tube) and porcine fetal-derived fibroblasts using the BD SMN antibody. A single band of approximately 38 kDa was detected in each cell type. HeLa cells served as a positive control. Actin was used as a loading control.

Analysis of porcine SMN in porcine tissues

Previous reports of western blot analysis have shown in human adult and fetal tissues that SMN protein is broadly expressed among tissue types but the level of SMN expression is regulated in a developmental and tissue-specific manner (Burlet et al., 1998). To examine tissue-type expression of SMN, we collected brain, heart, kidney, liver, muscle and spleen from wild-type, adult female and three 6 day old male swine (Figure 3A). In newborn animals, SMN protein appeared highest in the brain and spleen and SMN protein levels appeared lowest in the kidney and liver. In adult tissues, SMN protein was highly expressed in brain, kidney and spleen with lower levels of SMN detected in heart, liver and muscle. SMN levels declined in adult heart, muscle and spleen, and in the kidney and liver there appeared to be an increase of SMN from newborn to adult.

Fig. 3.

Fig. 3

Fig. 3

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Expression of SMN in porcine tissues. Brain, heart, kidney, liver, skeletal muscle and spleen tissues were collected from adult female and three 6 day old male swine. A: Expression of SMN protein in newborn and adult porcine tissues was analyzed by western blot by using the BD anti-SMN antibody. Actin was used as a loading control. Protein extracts were adjusted to the same total protein concentration before resolving by SDS-PAGE. B: Expression of SMN RNA in newborn and adult porcine tissues was analyzed by RT-PCR. RNA samples were adjusted to the same total RNA concentration before RT-PCR. Samples were analyzed using Multi Gauge (Fugi Film). The background sample was subtracted from each tissue sample and then the resulting number was normalized using the actin loading control. Primers to the porcine SMN1 cDNA were used for amplification. Porcine β-actin was used the control.

As there appeared to be differences in expression of SMN in different tissues from adult and newborn swine, we wanted to determine whether differences in SMN RNA expression were present (Figure 3B). Adult and newborn tissue from brain, heart, kidney, liver, muscle and spleen were analyzed by RT-PCR. In newborn piglets, SMN RNA was highest in the heart and spleen tissues and lowest in the kidney. In adult tissue SMN RNA expression appeared highest in the brain, liver and muscle and lowest in the heart and kidney. In adult heart and spleen there was a modest decrease in the amount of SMN RNA as compared to newborn with an increase in adult brain, liver and muscle (Figure 3B and Table 1). The relative ratio of newborn to adult SMN RNA was analyzed in these tissues. When the standard deviation of four independent amplifications was applied, there was only a slight difference in SMN RNA expression between newborn and adult in the tissues analyzed (Table 1).

Table 1. Analysis of SMN RNA expression.

The relative ratio of newborn to adult SMN RNA was determined using Multi Gauge (Fugi Film). Each sample was subtracted from background and normalized against β-actin. Error bars reflect the standard deviation. Four independent amplifications of porcine adult and newborn tissues were used in these studies

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Immunolocalization of porcine SMN protein to the cytoplasm and nuclear gems

SMN protein in human tissue-culture cells is detected diffusely in the cytoplasm and in distinct nuclear foci termed Gemini of Coiled Bodies or “Gems”. Gems are important biomarkers in SMA studies as there has been a correlation of gem number and disease severity established in primary patient fibroblasts. Many translational studies use increases in gem numbers as a measure of the “therapeutic effect” to increase SMN protein. To demonstrate that porcine SMN showed similar relevant localization patterns, we performed immunohistochemistry on fixed PK15, PFT-74 and fetal-derived fibroblast cells using an anti-SMN monoclonal antibody. HeLa cells, which contain high numbers of gems per nuclei, were used as a control. In each porcine cell type, a diffuse cytoplasmic staining and localization to distinct nuclear foci was detected (see arrows), consistent with gem localization (Figure 4).

Fig. 4.

Fig. 4

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Immunolocalization of SMN. Porcine cell lines PK15, PFT-74 and porcine fetal-derived fibroblasts were analyzed for distribution of SMN by using the BD anti-SMN antibody and a FITC-labeled anti-mouse secondary. SMN (green) localizes diffusely in the cytoplasm and to distinct nuclear foci (arrows). Nuclei were counterstained with DAPI (blue). Localization of SMN in porcine cells is consistent with that detected in HeLa as well as other human cells.

To determine whether there was a reciprocal relationship and therefore suggest that porcine SMN might function similarly as compared to human SMN, we transfected pEGFPC1-porcine SMN1a into HeLa, PK15 and fetal-derived fibroblasts (Figure 5). GFP expression was used to detect cells with transfected porcine SMN. The empty parental vector, pEGFPC1, served as a control (Figure 5). In PK15 and fetal-derived fibroblast cells, SMN from transfected pEGFPC1-porcine SMN1a exhibited subcellular patterns consistent with endogenous porcine SMN. In addition, porcine SMN localized similarly compared to endogenous SMN in HeLa cells. While these results cannot directly address the function of the porcine SMN protein, taken together, these results suggest that porcine SMN expression and localization is indistinguishable from human SMN.

Fig. 5.

Fig. 5

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Porcine SMN localizes in the cytoplasm and to nuclear gems in human and porcine cells. pEGFPC1-porcine SMN1a or pEGFPC1 was transfected into human HeLa and porcine PK15 and fetal-derived fibroblast cells. Expression of SMN was detected by GFP (green). GFP alone was used as a negative control. Expression of SMN from pEGFPC1-porcine SMN1a is consistent with that detected for endogenous porcine SMN and human SMN.

Porcine SMN1a and SMN1b differ slightly in their nucleotide sequence. The changes at positions 394 and 842 alter the coding sequence from asparagine to aspartic acid and lysine to arginine, respectively. The 3 nucleotide deletion results in the deletion of a proline in the exon 5 poly-proline region. To determine whether these nucleotide differences impacted expression or localization, pEGFPC1-SMN1a or pEGFPC1-SMN1b was transfected into HeLa, PK15 and porcine fetal-derived fibroblasts and GFP expression was examined. In all experimental contexts, these nucleotide substitutions did not impact SMN expression or localization in the cells tested (data not shown).

Co-localization of human and porcine SMN

Previous experiments demonstrated that the localization of porcine SMN was consistent with the localization of human SMN in human and porcine cells. To clearly demonstrate the co-localization of both proteins and the formation and localization of porcine SMN to nuclear gems, pEGFPC1-human SMN1 and pERFP-porcine SMN1b were co-transfected into porcine and human cells. Expression of human SMN was detected by GFP (green) and expression of porcine SMN was detected by RFP (red). In every cell type tested, human and porcine SMN demonstrated co-localization in the cytoplasm and in nuclear gems (Figure 6 and data not shown).

Fig. 6.

Fig. 6

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Porcine and human SMN co-localize. pERFP-porcine SMN1b and pEGFPC1-human SMN1 were co-transfected into porcine PK15 cells. Expression human SMN was detected by GFP (green) and porcine SMN was detected by RFP (red). Both human and porcine SMN co-localize diffusely in the cytoplasm and to nuclear gems (yellow in merge). Nuclear gems are indicated with arrows. Nuclei were counterstained with DAPI (blue).

Porcine SMN1 compensates for a deficiency in human SMN1

Type I is the most severe form of Spinal Muscular Atrophy. Primary fibroblasts derived from a Type I SMA patient (3813) have a substantial reduction of SMN protein and contain 3-8 gems/100 nuclei while fibroblasts derived from a non-affected carrier parent (3814) typically contain ∼52 gems/100 nuclei (Figure 7A and D). These cells were used to determine whether porcine SMN could rescue the severe reduction in SMN protein and the loss of gem formation in 3813 fibroblasts. 3813 cells were transiently transfected with pEGFPC1 alone, pEGFPC1-porcine SMN1 or pEGFPC1-human SMN1. In all transfected 3813 fibroblasts, transfection of porcine SMN1 restored the formation of SMN-containing gems (Figure 7B). These results were consistent with those obtained with transfection of pEGFPC1-human SMN1 (Figure 7C). Untransfected and pEGFPC1 transfected 3813 fibroblasts served as controls (Figure 7B). To determine whether there was an increase in total SMN protein, 3813 type I fibroblasts were transiently transfected with pEGFPC1-porcine SMN1. Cell lysates were analyzed by western blot using the human anti-SMN antibody to detect endogenous and transfected SMN. In these studies, a substantial increase in SMN protein above untreated 3813 fibroblasts was detected (Figure 7D). The increase in endogenous SMN protein may be attributed to the stabilization of SMN protein due to SMN’s ability to self-associate with GFP-SMN following transient transfection (Figure 7D). Untreated HeLa cells which contain high levels of SMN as well as unaffected 3814 carrier fibroblasts were used as positive controls (Figure 7D). Actin was used as a loading control. These studies suggest that porcine SMN can form SMN-containing gems in human SMA type I fibroblasts that are severely deficient of gems and increase the total SMN protein in these cells.

Fig. 7.

Fig. 7

Fig. 7

Fig. 7

Fig. 7

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Porcine SMN1 compensates for a deficiency in human SMN1. A: SMN expression in 3814 non-affected carrier fibroblasts and 3813 SMA type I patient-derived fibroblasts. SMN (green) localizes diffusely in the cytoplasm and to distinct nuclear gems (arrows) in 3814 cells; however, formation of SMN-containing gems is nearly absent in 3813 fibroblasts. B: 3813 patient-derived fibroblasts transfected with pEGFPC1-porcine SMN1a form SMN-containing nuclear gems. Untransfected and 3813 cells transfected with pEGFPC1 serve as controls. C: 3813 patient-derived fibroblasts transfected with pEGFPC1-human SMN1 form SMN-containing gems consistent with the results observed using porcine SMN1. Nuclei were counterstained with DAPI (blue). D: Total SMN protein increases in 3813 patient-derived fibroblasts following transfection of pEGFPC1-porcine SMN1a. Untreated 3813 SMA type 1 fibroblasts contain reduced levels of SMN protein as compared to the non-affected 3814 carrier fibroblasts. HeLa cells, which contain high levels of SMN, were used as a control. Actin was used as a loading control.

DISCUSSION

We have identified, cloned and characterized two porcine SMN1 cDNAs. The SMN open reading frame is 882 (SMN1a) or 885 nts (SMN1b) and encodes a protein of 293 (294) amino acids respectively, with a molecular weight of ∼38 kDa. SMN1a contains A to G alterations at positions 309, 394, 842 and 864 and a 3 nucleotide ACC deletion at 669-671. Interestingly, both G nucleotides in exon 7 of SMN1a (842 and 864) are also found in human SMN1. The changes at 394 and 842 alter the coding sequence; however, these nucleotide differences do not appear to alter the expression or localization of the SMN1a or SMN1b tagged constructs in the cells tested. Additionally, the single proline deletion in the SMN1a cDNA localizes to the cytoplasm and to nuclear gems as does SMN1b and human SMN1. The loss of a single proline in the poly-proline symmetrical Pro5-X17-Pro10-X17-Pro5 motif (for SMN1a) would likely have little impact as there is slight variation between species with mice having 11 poly-prolines, humans and rats 10 and bovine 9 in the Pro10 motif. The only role to date associated with poly-proline motifs and SMN is the interaction of the microfilament associated proteins, Profilins (Giesemann et al., 1999).

Analysis of the mutation spectrum in patients with SMA showed that most (∼92%) SMA patients carry a homozygous deletion of the SMN1 gene. However, there are approximately 30 different subtle intragenic mutations identified (Wirth, 2000; Sun et al., 2005). These subtle mutations account for approximately 3.4% of SMA patients. Y272C located in the exon 6 self-association domain is the most frequent occurring subtle mutation (20%). In fact, most missense mutations are localized to exon 6 and show decreased self-oligmerization (Lorson and Androphy, 1998; Wirth, 2000). Interestingly, exon 6 is 100% conserved between swine and human and a comparison of the predicted proteins of human, cow, swine, mouse and rat show 100% conservation of those specific amino acids with significant conservation found in the fruit fly and worm. Therefore, it is not surprising multiple mutations in exon 6 are associated with SMA disease formation. Several other missense mutations, including E134K, G95R and A111G in the Tudor Domain have been associated with complete or reduced capacity to bind Sm proteins (Buhler et al., 1999; Sun et al., 2005). Sequence conservation and mutation analysis emphasizes the relevance of these amino acids in disease formation.

In Sus scrofa we find tissue-specific and developmental-specific SMN protein expression consistent with that observed in human and mouse tissues. SMN protein is highest in the brain of newborn piglets with high expression also detected in the heart and spleen. There is a remarkable decrease in SMN expression from newborn to adult in heart, muscle and spleen. However, there is not a reciprocal dramatic change in SMN RNA from newborn to adult in any of the tissues tested.

The localization of porcine SMN to the cytoplasm and to nuclear gems is in agreement with SMN functioning in snRNP biogenesis. We have shown that transfection of human SMN1 into porcine cells results in the expression and correct localization of human SMN. In a reciprocal experiment, porcine SMN1 was transfected into human HeLa cells resulting in the expression and expected localization of porcine SMN. In addition, we have demonstrated that porcine SMN can compensate for the deficiency of human SMN in 3813 SMA type 1 fibroblasts by increasing total SMN protein and the formation of SMN-containing nuclear gems. These studies, as well as the highly conserved sequence between human and swine SMN suggest that SMN interacting proteins are present in porcine cells and that porcine SMN can interact with SMN complex members in human cells.

These initial studies are important as they provide the foundation for future experiments demonstrating conserved function and the ability of porcine SMN to functionally compensate for human SMN. The conservation in sequence, localization and expression suggest that the development of a SMA transgenic swine is possible. A larger SMA animal will provide an excellent experimental model to begin to systematically address questions of the efficacy of candidate therapeutics, including whether prospective SMA therapies reach their motor neuron targets and what levels of full-length SMN are present in motor neurons following such treatments. As well, the scale of the swine nervous system provides a better “tool” for the examination of retrograde transport following viral gene transfer in order to better understand the most efficient means to deliver gene therapies.

Experimental Procedures

Genomic Sequence

Porcine genomic DNA was isolated from PK15 and fetal-derived fibroblast cells using the Purelink Genomic DNA Purification Kit (Invitrogen). Introns were amplified using primers to the adjacent exons. PCR products were cloned into pCRII-Topo (Invitrogen) and multiple clones were bi-directionally sequenced. Overlapping clones from exons 2b through 4, 3 through 5 and 4 through 6 were also cloned and sequenced.

Cloning of the porcine SMN1 cDNA

PK15 and fetal-derived fibroblast cells were harvested and washed 2X in PBS. Cells were then subjected to a TRIzol (Invitrogen) extraction and RNA was isolated. First-strand cDNA synthesis was performed by using Super Script III (Invitrogen) at 42°C for 50 minutes followed by 70°C for 10 minutes. Two microliters of the first-strand synthesis was used in the subsequent PCR amplification [94°C for 5 min, (94°C for 0:45 min, 55°C for 1 min, 68°C) X 35] by using Accuprime Pfx Polymerase (Invitrogen). The PCR product was gel purified and sequenced. Upon confirming the sequence, the porcine SMN1 cDNA was cloned inframe into pET32A (Novagen) and pEGFPC1 (BD Biosciences) using primers with engineered restriction sites. Positive clones in each expression vector were identified by colony PCR and sequenced. This cDNA is identified as SMN1a. A second SMN1 cDNA identified as the TIGR consensus sequence for TC239547 was synthesized by GENEART, cloned in-frame into pEGFPC1 and pERFP (BD Biosciences) and sequenced. This cDNA is identified as SMN1b.

Western blot analysis

Porcine tissues where harvested from a wild-type female adult and three 6 day old male Sus scrofa and then frozen in liquid nitrogen. Tissues were homogenized in a protease cocktail (50 mM Tris-HCl, 10 mM EGTA, 5 mM EDTA, protease inhibitor), incubated on ice for 30 minutes, sonicated and incubated on ice for 15 minutes. Protein concentrations were determined by using the Bradford Assay (Bio Rad) with BSA as a standard. Lysates were boiled for 5 minutes and resolved on a 10% SDS-PAGE gel. Experiments for each sample were performed in triplicate.

Cells from HeLa (human cell line), PK15 (porcine kidney cell line), PFT-74 (porcine fallopian tube cell line), porcine fetal-derived fibroblasts, 3813 SMA Type 1 patient-derived fibroblasts and 3814 non-affected carrier-derived fibroblasts were collected, washed 3X in PBS and resuspended in 5X loading dye. Lysates were boiled for 5 minutes, sonicated and resolved on a 10% SDS-PAGE gel.

Membranes were blocked in 5% Nonfat Dry Milk overnight, washed in TBST (Tris Buffered Saline with Tween) followed by incubation with the primary antibody for 1 hour at room temperature. Subsequently the membrane was washed 3X for 15 minutes each in TBST and reacted with the secondary antibody at room temperature for one hour. Membranes were developed by chemiluminescence using Pierce SuperSignal Pico reagents. Antibodies used for these studies included anti-SMN mouse monoclonal antibody (BD Biosciences 1:500) and an anti-actin polyclonal rabbit antibody (Sigma 1:400).

RT-PCR analysis of porcine tissues

Porcine tissues where harvested from wild-type female adult and 6 day old male Sus scrofa and then frozen in liquid nitrogen. Tissues were homogenized in TRIzol (Invitrogen) and then subjected to a TRIzol extraction and RNA was isolated. 2 μg of RNA were used for each first-strand cDNA synthesis. First-strand cDNA synthesis was performed by using Super Script III (Invitrogen) at 42°C for 50 minutes followed by 70°C for 10 minutes. One microliter of the first-strand synthesis was used in the subsequent PCR amplification [94°C for 3 min, (94°C for 30 sec, 56°C for 1 min, 72°C for 1 min) X 25] by using EconoTaq (VWR). Porcine β-actin was used as a loading control. Four independent PCR amplifications were carried out on one adult female and three newborn males. Samples were analyzed by using Multi Gauge (Fugi Film). The background sample was subtracted from each tissue sample and then the resulting number was normalized using the actin loading control to obtain a normalized tissue sample.

Immunohistochemistry

The following cell lines were used for immunohistochemical analysis: HeLa, PK15, PFT-74, 3813 SMA Type 1 patient-derived fibroblasts and 3814 non-affected carrier-derived fibroblasts. Porcine fetal-derived fibroblast cells were isolated as previously described (Lai et al., 2002). Cells were trypsinized, and plated at 70-80% confluency in 60 mm dishes containing coverslips. Cells were fixed with a 1:1 methanol/acetone solution for 5 minutes and left to air dry. Cells were blocked in 5% BSA (Bovine Serum Albumin) in PBS (Phosphate Buffered Saline-Gibco) for 1 hour at room temperature; washed 3X for 15 minutes each in TBST, and reacted with the primary antibody overnight at 4°C. Coverslips were washed 3X with TBST for 15 minutes each at room temperature, incubated with secondary antibody for 1 hour at room temperature, and rewashed. One ug/ml DAPI was added to the coverslips for 5 minutes and the coverslips were mounted onto slides using 1% DABCO mounting media. Antibodies used for these studies were: human anti-SMN (BD Biosciences). Antibodies were diluted in a solution containing 1% BSA in PBS. Proteins were visualized by using FITC-conjugated secondary antibody (Sigma, 1:200).

Transfections

Subconfluent HeLa, PK15, 3813, 3814 or porcine-derived fetal fibroblast cells were plated on 60 mm dishes with coverslips and transfected with pEGFPC1, pEGFPC1-porcine SMN1a, pEGFPC1-porcine SMN1b, pERFP-porcine SMN1b or pEGFPC1-human SMN1 by using Lipofectamine 2000 (Invitrogen) or PEI (polyethyleneimine). Transfected cells were fixed 24 hours post-transfection by using 1:1 methanol/acetone as described above.

Supplementary Material

Supp Data
Supp Fig

ACKNOWLEDGEMENTS

MAL would like to especially thank Dr. Kevin Wells and members of the Prather laboratory for their kindness and assistance with these studies. Thanks also to members of the C. Lorson laboratory for their support of this project. This work was funded by grants from the College of Veterinary Medicine (University of Missouri) and FightSMA to MAL. CLL is supported by the National Institutes of Health (R01HD054413 and R01NS041584). RSP is supported by the National Institutes of Health (RR13438) and Food for the 21st Century.

Grant Information: FightSMA to M. Lorson, College of Veterinary Medicine to M. Lorson, NIH R01HD054413 to C. Lorson, NIH R01NS041584 to C. Lorson, NIH RR13438 to R. Prather, Food for the 21st Century to R. Prather

GeneBank Accession Numbers

Human SMN protein: AAC652048, Mouse SMN protein: AAD56757

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Supplementary Materials

Supp Data
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Supp Fig
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