Genetic correction of splice site mutation in purified and enriched myoblasts isolated from mdx5cv mice
© Maguire et al; licensee BioMed Central Ltd. 2009
Received: 10 November 2008
Accepted: 23 February 2009
Published: 23 February 2009
Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disorder that results in the production of a dysfunctional form of the protein, dystrophin. The mdx5cv mouse is a model of DMD in which a point mutation in exon 10 of the dystrophin gene creates an artificial splice site. As a result, a 53 base pair deletion of exon 10 occurs with a coincident creation of a frameshift and a premature stop codon. Using primary myoblasts from mdx5cv mice, single-stranded DNA oligonucleotides were designed to correct this DNA mutation.
Single-stranded DNA oligonucleotides that were designed to repair this splice site mutation corrected the mutation in the gene and restored expression of wild-type dystrophin. This repair was validated at the DNA, RNA and protein level. We also report that the frequency of genetic repair of the mdx mutation can be enhanced if RNAi is used to suppress expression of the recombinase inhibitor protein Msh2 in cultures containing myoblasts but not in those heavily enriched in myoblasts.
Exogenous manipulations, such as RNAi, are certainly feasible and possibly required to increase the successful application of gene repair in some primary or progenitor muscle cells.
Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder with an incidence of 1 in 3,500 live born males. It is caused by the dysfunction of dystrophin (DMD), a large 427 kDa cytoplasmic protein that forms a link between actin filaments and the dystrophin-glycoprotein (DGC) complex . The loss of dystrophin is thought to affect the integrity of the cell membrane leading to a disruption in cellular calcium homeostasis and eventually cell necrosis [1–3]. At the genetic level, one-third of boys afflicted with DMD have point mutations, insertions or deletions while the remaining two-thirds have gross deletions or rearrangements within the 2.5 Mb gene  [OMIM #310200 Muscular Dystrophy, Dystrophy Type, DMD] and all generally cause a translational frameshift.
Therapeutic strategies such as gene replacement have been used to restore protein expression by effectively re-engineering dystrophin cDNA. In addition, attempts have been made to introduce smaller functional homologs of DMD to cells in an attempt to reverse the muscle degradation process [5–8]. Vectors, which carry these constructs, however, often elicit an immune response [8, 9]. An RNA/DNA chimeric oligonucleotide (chimera) has been used successfully to correct the splice site mutation to generate a genetic alteration in the dystrophin gene, in both ex vivo and in vivo studies in mouse and dog models of DMD [10–13]. In a pioneering study, Bartlett et al.  showed the correction of the canine dystrophin gene within skeletal muscle fibers after injecting the chimera into the cranial tibialis of Golden Retrievers. The production of wild-type dystrophin was restored and could be detected for up to 48 weeks at the site of the injection. Rando et al.  demonstrated chimera-directed repair of the dystrophin gene in the mouse extending the results from the dog model; once again the corrected cells were again localized to the injection site.
Antisense oligonucleotides also have been used to alter the splicing of the dystrophin message in a process generally termed, exon skipping [14–16]. A large number of exciting reports have demonstrated the feasibility of altering the splicing patterns of the mutant DMD transcript [15, 17–21]. While this approach is likely to have significant therapeutic benefit, the original DMD mutation(s) remains and thus a continuum of antisense ODN delivery will be required .
When muscle fibers are damaged within the body, a population of quiescent precursor muscle cells, or satellite cells, become activated myoblasts [23, 24], rapidly divide and initiate the regeneration of the injured muscle . In the case of DMD, the muscle is thought to be constantly going through cycles of degeneration and regeneration due to the instability of the musculature and, therefore, abundantly populated with myogenic precursor cells. Thus, an important long term therapeutic approach then would be to isolate and genetically correct the myoblasts at the DNA level with subsequent transplantation to the animal model in order to restore functional dystrophin to the muscle fibers.
We have begun to develop a more defined ex vivo strategy in which modified single-stranded oligonucleotides are designed to repair the mutant dystrophin (ex vivo) in cell cultures enriched in myoblasts. A current challenge is to establish a frequency of repair that leads to a level of dystrophin expression that can be detected in targeted myoblasts. Previously, Bertoni et al.  have used fluorescently labeled oligonucleotides to correct a point mutation in cells derived from the dystrophic mdx5cv mice. We now repeat the correction of Dmd point mutation using unlabeled modified single stranded oligonucleotides and establish novel quantitative assays to measure the dystrophin production in myoblasts. We also stimulate the correction reaction by using RNAi against Msh2, a known inhibitor of the gene repair reaction  and observe higher levels of gene repair.
Isolation of primary cultures from dystrophic mdx5cv mice containing myoblasts with determination of isolation efficiency
In the natural muscle environment, satellite cells rest in a quiescent state along the periphery of an intact muscle fiber. When muscle fibers are damaged, muscle precursor cells, referred to as myoblasts, divide and fuse with the damaged muscle fibers to repair them [see  for review]. In order to isolate the muscle precursor cells for gene repair experiments, muscle tissue was removed from the limbs of 1 to 3 days old mdx5cv mice, enzymatically digested and plated. A pre-plating technique was used to purify these cells from fibroblasts present in the explant [27, 28]. It is known that the fibroblasts adhere to the plate rapidly, usually within 2 hours, and, therefore, the pre-plating technique is used them from the culture by passaging the supernatant at a 2 hour time point (PP1) and again at a 24 hour time point (PP2) . Thus, the supernatant is passaged until PP4 at which time cells are harvested to determine the efficiency of myoblast isolation by incubation with an antibody against desmin (expressed in myoblasts). We estimate the efficiency of myoblast isolation in this experiment to be 53% as determined by counting the number of desmin positive cells and dividing this number by the number of total cells observed in multiple fields of view (data not shown). The remaining 47% are most likely fibroblasts. Thus, in the experiments outlined below, we utilize cultures that contain approximately 50% myoblasts and 50% fibroblasts. Primarily myoblasts are a target because only corrected myoblasts that fuse to form myotubes will be capable of expressing corrected dystrophin.
Gene repair of dystrophin directed by modified single-stranded oligonucleotides in purified myoblasts from mdx5cv mice
We outlined oligonucleotide-mediated gene repair reactions containing ODNs, mdx47NT or mdx47T, designed to correct the T substitution mutation within exon 10 of the dystrophin gene. These are depicted in Figure 2. For clarity, we provide the wild type and mutant DNA sequence, as well. The "T" indicates that the 47-mer is designed to target the transcribed strand while the "NT" refers to a molecule designed to target the non-transcribed strand.
Quantitative measurement of dystrophin, GAPDH, and CKM.
34.1 ± 0.15
33.8 ± 0.22
34.9 ± 0.08
37.1 ± 0.35
13.2 ± 0.08
13.6 ± 0.02
13.3 ± 0.05
13.6 ± 0.32
22.1 ± 0.11
13.6 ± 0.11
21.5 ± 0.08
13.6 ± 0.10
Enhancement of dystrophin correction by knockdown of Msh2
Successful correction of the dystrophin gene using either the RNA/DNA chimera or the modified single stranded oligonucleotide has been achieved in both dogs and mice by direct injection of synthetic DNA molecules [10–12, 25]. The frequency of correction throughout muscle tissues however must be increased to enable therapeutic benefit. Bertoni et al.  have demonstrated that when muscle is injected directly with synthetic oligonucleotides, genetic correction was restricted to the dystrophin gene within a population of satellite cells. Since the frequency of gene repair is higher when delivery to cells outside the body takes place in an ex vivo approach. Thus, a more efficient approach for the treatment of DMD might entail using ex vivo delivery with subsequent transplantation back into the host.
To enable this ex vivo strategy, it was essential to optimize the efficiency of ODN delivery and ODN-mediated correction of myoblasts. This was accomplished by transfecting a culture of primary muscle cells enriched in myoblasts with a FAM labeled oligonucleotide and measuring uptake by FACs analysis. We confirmed that the ODN was present in the nucleus by examining the nuclei with confocal imaging (data not shown). Correction of the point mutation in the dystrophin gene was then demonstrated at the level of DNA, RNA and protein. Analysis of the quantity of corrected DNA indicated that the correction frequency remains stable over the course of 96 hours when targeting with either mdx47NT or mdx47T. A significant increase in message containing the portion of exon 10 deleted in the mutant cells was also detected at both 48 and 72 hours after transfection. There was no reduction in the level of repaired RNA over time which suggests that the corrected gene produces stable and inheritable message.
DNA analyses indicate that correction occurs at a 3 fold higher frequency when the nontranscribed strand of the gene is targeted. One hypothesis for strand bias is that the movement of RNA polymerase would interrupt the pairing of the ODN and this would result in lowered correction efficiency . The dystrophin gene, however, is expressed only in differentiated cells and therefore, ODN binding stability should not be disrupted by the movement of the RNA polymerase. Alternatively, the orientation of the gene in terms of the general direction of replication fork movement can influence the targeting frequency in that the lagging strand targets at a higher level than the leading strand . Thus, the strand designated as nontranscribed may also be the lagging strand at the target site.
Previous experiments of Ferrara and Kmiec  show that the corrected cells reduce their cell division cycle after introduction of the oligonucleotide because the free DNA ends activate G2 checkpoint proteins. During this time, the uncorrected population is able to divide, which will, in effect, cause the number of corrected cells to be diluted in number. This results in a lower measure of correction efficiencies as division in the uncorrected population is not impaired. In primary myoblasts cultures, however, this does not seem to be the case as the whole population of cells continues to divide with the correction efficiency maintained. This observation suggests that the corrected cells do not suffer a growth disadvantage by this ex vivo protocol.
Duchenne Muscular Dystrophy (DMD) is caused by an absence of the protein dystrophin as indicated by the absence of immunostaining for this protein in most of the muscle fibers. We observed elevated levels of dystrophin in cells that were treated with specific ODNs designed to correct the mutated dystrophin gene and after introduction of the oligonucleotide, myotubes that are expressing a significant amount of dystrophin are detected at 72 hours. The number of corrected cells that contribute dystrophin to the positive myotubes is unknown, but it is interesting to note that each myotube that stained positive had dystrophin evenly distributed throughout. This indicates that the dystrophin produced by the corrected gene can diffuse throughout a small myotube in vitro and would probably diffuse though part of a myofiber in vivo, in a region called the nuclear domain . The long-term goal of the present study is to transplant corrected myoblasts back into mice to restore dystrophin production in muscle fibers. To achieve this goal it was necessary to establish ex vivo targeting conditions wherein the highest level of correction frequency in purified cells is achieved. The observation that Msh2 knockdown enhanced the repair frequency also indicates that the mechanism of gene repair is conserved in primary cells and has, to some degree validated basic studies in established model systems . To our surprise, RNAi knockdown of Msh2 in cultures of purified myoblasts did not lead to an elevation in the repair of the mutant dystrophin gene, regardless of increasing amounts of correcting ODN at the varying doses of RNAi. The overall level of correction in the purified cultures is substantially lower and may be at a basal level where adjuvant treatments designed to stimulate activity do not impact the gene repair reaction. In addition, these results suggest that the stimulation of correction in the enriched cultures was likely taking place in the fibroblasts which contaminate the culture. For unknown reasons, primitive progenitor cells are less amenable to gene repair or at least provide an environment, less conducive for the reaction.
Correction levels vary among cell types with certain mutant targets being repair at levels approaching 1–5%. Yet, the consensus view is that progenitor cells, at any step along the differentiation pathway, do not enable this reaction to take place efficiently [39–41]. A series of natural barriers seems to exist in these cell types including lower transfection efficiencies and dosage-dependent toxicity. Increasing the amount of ODN introduced into the cell usually results in a gradual increase in side effects . Progenitor cells are usually more sensitive to the transfection agent  and strand bias for targeting can also vary widely based in gene types . Murphy et al.  found a mismatch hierarchy that contributed to the various levels of gene correction; A:G > A:A > A:C. If this is true, then the capacity to target progenitor cells will be hampered. Such a hierarchy has not been observed in somatic cell targeting where correction efficiencies are generally higher.
Thus, progenitor cells will present unique challenges that will require exogenous manipulation to elevate frequency of gene repair. While MSH2 knockdown appears not to be a sufficient protocol to meet this goal in myoblasts, it does not mean that other progenitor lineages will not response positively. In fact, Dekker et al.  have already shown that MSH2 knockdown does increase the frequency of gene repair in mouse ES cells. More recently, Aarts et al.  provided support and confirmed these observation and Maguire and Kmiec  extended this protocol successfully into mouse embryonic fibroblasts. Morozov and Wowrousek  followed among the same theme with observations that inhibition of Rad51/54 and Ku70/86 led to an increase in gene correction efficiency. These types of cellular manipulations are certainly feasible and may be required for the successful application of gene repair in primary or progenitor cells.
Cells and oligonucleotides
Primary myoblasts derived from the mdx5cv mice were maintained in DMEM supplemented with either 10% or 20% FBS. When the cells were induced to differentiate, DMEM containing 2% horse serum (DM) was added to the cells for at least 96 hours. The mutation of the parental plasmid pEGFP N3 to create the mutant pEGFP N3 (rep) has been described previously . Targeting oligonucleotides, eGFP3S/47NT, mdx47NT, mdx47T, and mdx47NT-FAM were obtained either from Sigma Genosys (St. Louis, MO) or IDT (Coralville, IA) where they were synthesized and purified by HPLC before distribution. All modified single-stranded oligonucleotides were resuspended to a concentration of 200 μM and aliquots were stored at -20°C. The mdx5cvmice were obtained from the Jackson Laboratory.
Isolation of myoblasts from newborn mdx5cv mice
The isolation procedure for mouse myoblasts and subsequent pre-plating methods have been described previously [27, 28]. This work was authorized by the Laval University Animal Care Committee and experiments were conducted in accordance with guidelines set forth by the Canadian Council of Animal Care. Briefly, limbs of 1 to 3 days old male mdx5cvmice were isolated and muscle was pulled from the bone and cartilage using sterile forceps. The muscle was treated for 1 hour with Collagenase (Sigma, St. Louis, MO) and Dispase II (Roche, Indianapolis, IN) to degrade collagen and to disrupt cell contacts. The cells were plated for 2 hours to allow fibroblasts, which rapidly adhere, to attach to the petri dish (PP2). The supernatant, containing a mixture of myoblasts and fibroblasts, was removed and the cells are passaged again to another petri dish (PP3). After 24 hours the cells were again passaged (PP4) and subsequently transferred to another culture dish (PP5). Purified myoblasts were expanded and used in gene repair experiments.
Cells were aliquoted into 4 wells of an 8 well chamber at a density of 1 × 105 cells, and allowed to attach overnight. The cells were fixed using 100% ethanol for 5 minutes at -20°C, permeabilized with 0.1% Triton X-100, and then incubated with either desmin (Sigma, St. Louis, MO) specific or dystrophin specific antibodies raised in rabbit (Abcam, Cambridge, MA 1:1,000) at room temperature (RT) for 1 hour or overnight at 4°C. After washing the cells, a secondary goat α-rabbit antibody conjugated to a Cy3 fluorophore (desmin) (Zymed Laboratories, San Francisco, CA) or an Alexa 546-coupled goat anti-rabbit antibody (Molecular probes 1:1500) was added to the wells and this mixture was incubated with agitation for 1 hour at room temperature. After four 15 minute washes, the wells were dried and mounting media containing DAPI was added to each well (Molecular Probes, Eugene, OR). The cells were then visualized using a Zeiss™ inverted 100 M Axioscope equipped with a Zeiss™ 510 LSM confocal microscope with a coherent krypton argon laser (30 mW) containing 488 nm or 568 nm and 742 nm excitation lines.
Gene repair of dystrophin in purified myoblasts
Cells were plated in 6-well culture dishes at a density of 1 × 105 with media containing 20% FBS. jetPEI was used to make oligonucleotide complexes with 3 μg of ODN at an optimal N/P ratio (nitrogen/phosphate ratio) of 5. This reagent was added to 1 ml of media and the cells were incubated for various amounts of time at which point differentiation media (DM) was added; the cells were differentiated for 96 hours, then harvested for FACS analysis and genomic DNA isolation. For mRNA analyses, the cells were collected at least four days after the addition of DM containing 2% horse serum.
Knockdown of Msh2
RNAi, designed to knock down Msh2, was purchased from Ambion (Ambion, Austin, Texas); siRNA (ID# 155431) is designed to anneal to the mouse MSH2 RNA at exon 3 NM_008628. During the targeting experiments, the RNAi was mixed with the correcting oligonucleotides complexed with jetPEI and added to the cells as described above.
Co-transfection ssODNs and Msh2 RNAi
Cells were plated in 6-well plates at a density of 1 × 105 cells/well with growth media (DMEM supplemented with 20% FBS, penicillin and streptomycin). Twenty four hours after plating, cells were co-transfected 3 μg of mdx/47NT and RNAi specific to knockdown Msh2 (0–200 nM). Seventy two hours after transfection, the growth media were replaced by differentiation media (DMEM supplemented with 2% horse serum, penicillin and streptomycin), and the cells were maintained in differentiation media for 96 h. Total RNA was isolated from the cells, and reverse transcription and quantitative PCR were conducted as described in "RNA isolation and, RT PCR and quantitative PCR.
Cells were plated in 6-well plates at a density of 1 × 105 cells/well with growth media. Twenty four hours after plating, cells were co-transfected 3 μg of mdx47NT and RNAi specific to knockdown Msh2 (1–100 nM). Total RNA was isolated 3 or 6 h after transfection Methods of reverse transcription and quantitative PCR were same as "RNA isolation and, RT PCR and quantitative PCR", except for primers: musMSH2/257F 5' agaccctgcagagtgttgtgctta 3'; musMSH2/1010R 5' tgagagccagtggtgtcttcaaca 3' or mouse GAPDH4FwQ 5' tgtctcctgcgacttcaacagc 3'; GAPDH5RevQ 5' atgtaggccatgaggtccacca 3'.
RNA isolation, RT PCR and quantitative PCR
After differentiation of the myoblasts, the cells were harvested by trypsinization and collected by centrifugation at 1,500 rpm for 7 minutes. After washing with 1× PBS, the RNA was extracted using a Qiagen RNA extraction kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Three hundred nanograms of RNA was subjected to reverse transcription to generate cDNA using a gene specific primer Rev-ex12 5' cag gtc caa agg gct ctt cc 3' or primer oligo dT using the first strand synthesis kit (Invitrogen, Carlsbad, CA). Five microliters was added to a 20 μL mixture of 2× Sybrgreen Master Mix (Applied Biosystems, Foster City, CA) with 2.5 μM of each primer:
Forw-ex 9 5' gtt atg cct tca cag gct gc 3'; Rev-ex10 in 5'cacttc ttc aac atc att tg3' or GAPDH4FwQ 5'tgtctcctgcgacttcaacagc3'; GAPDH5RevQ 5'atgtaggccatgaggtccacca 3' CKM F1 5' GCAACACCCACAACAAGTTCA 3' and CKM R1 5' TGTCTCCTTATCGCGAAGCTT 3'. Samples were added to each well of a 96 well plate in duplicate for each primer set. Dystrophin was amplified using the cDNA generated by the gene specific primer and GAPDH or CKM from cDNA generated using oligo dT. QPCR was run for 1 repetition at 50°C for 2 minutes, 1 repetition at 95°C for 10 minutes, 40 repetitions at 95°C for 15 seconds and 40 repetitions at 59°C for 1 minute with data collection at step 1 of stage 3. Upon completion of PCR, the threshold and baseline values were set to measure Ct values at the beginning of the exponential amplification. Ct values from 3 experiments were averaged per sample and subjected to a student's t-test and ANOVA to determine significance.
DNA isolation and absolute quantification by PCR
Cells were harvested and collected by centrifugation at 1500 rpm for 7 minutes and washed twice with 1× PBS or lysed directly in a tissue culture dish after washing with 1× PBS. DNA was precipitated using the genomic wizard DNA extraction kit as instructed by the manufacturer (Promega, Madison WI). Twenty-five nanograms of genomic DNA was added to a total of 7 μL of water and this was mixed with 13 μL of a 2× PCR master mix, 50 nM of a 6FAM labeled probe specific to bind to wild-type dystrophin 5'FAMcac aag gag aga ttt3', 900 nM of forward primer mdx5cvasF 5' ttt ctg ccg agg ata cat tgc3' and 900 nM of reverse primer ex 10 out 5' ctc atg agc atg aaa ctg ttc 3'. Each experimental protocol was run in triplicate. A standard curve was generated using 50 ng, 25 ng, 12.5 ng, 6.25 ng, and 3.13 ng of genomic DNA isolated from C2C12 cells, which are wild-type for dystrophin. As a separate control, reaction mixtures containing 25 ng of genomic DNA from mdx5cv were used to determine the background of wild-type DNA in each experiment. PCR conditions were: 1 repetition at 50°C for 2 minutes, 1 repetition at 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The data were collected at stage 3 of step 1. Threshold and baseline levels were set to measure the Ct values at the lowest level of exponential amplification and the average mean was determined from each experiment. Statistical significance was determined by a student's t-test and was considered significant only if p was less than 0.05. Following the PCR amplification the products were purified using Qiagen's QIAquick PCR purification kit according to the instructions and submitted for sequencing using the mdx5cvasF primer.
Primary mouse myoblast purification
The enriched culture obtained from Tremblay lab was plated on a collagen-coated culture dish with F10 nutrient mixture (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) FBS, 2.5 ng/ml bFGF (Promega, Madison, WI), 100 U/ml penicillin and 100 mg/ml streptomycin. When the culture reached confluency, cells were detached from the dish without using trypsin. To accomplish this, the dish was washed with PBS after aspiration of medium, incubated with a small amount of PBS at room temperature for 5 min, and hit firmly sideways. Predominant cell type in the detached cells was myoblast due to the fact that myoblasts are likely to detach from collagen-coated dish than fibroblasts are. The detached cells were collected with F10 nutrient mixture, plated on a new collagen-coated dish, and expanded. The above procedures were repeated until no fibroblasts were observed in the culture. Upon completion of purification, medium was switched F10 growth medium to F10/DMEM (1:1) supplemented with 10% (v/v) FBS, 2.5 ng/ml bFGF (Promega, Madison, WI), 100 U/ml penicillin and 100 mg/ml streptomycin.
Cells were plated on 2-well chambers at a density of 8 × 104 cells/well and incubated at 37°C overnight. The cells were fixed using ethanol for 5 minutes at -20°C, permeabilized using 0.3% (v/v) TritonX for 10 minutes at room temperature, blocked using 5% (v/v) heat-inactivated goat serum for 30 minutes at room temperature, and then incubated with desmin specific antibody (Sigma, St. Louis, MO; dilution, 1:50) for 1 hour at room temperature. After washing the cells, a secondary goat a-rabbit antibody conjugated to Cy3 (Zymed laboratories, San Francisco, CA; dilution, 1:750) was added to the wells and this incubated with agitation for 1 hour at room temperature. After four 15 minute washes, the wells were briefly dried and mounting media containing DAPI was added to each well (Molecular Probes, Eugene, OR).
Oligo dose experiment
Purified myoblasts were plated in 6-well plates at a density of 1.5 × 105 cells/well with F10/DMEM growth medium 24 hours before transfection. jetPEI was used to make oligonucletide complexes with various amounts of mdx47NT at N/P (nitrogen/phosphate) ratio of 5. This reagent was added to 1 ml of media and the cells were incubated. Genomic DNA was isolated 24 hours after transfection using masterpure DNA purification kit (Epicentre, Madison, WI). TaqMan® probe-based real time PCR was performed to quantify wild type dystrophin. Probe specific for wild type dystrophin (5'-FAMcac aag gag aga ttt-3') were used with the primer pair: forward primer, 5'-ttt ctg ccg agg ata cat tgc-3'; reverse primer, 5'-ctc atg agc atg aaa ctg ttc-3'. Real time PCR was performed with 25 ng genomic DNA, 900 nM both forward and reverse primer, and 250 nM probe in each 20 ml reaction. A standard curve was generated using 1, 0.5, 0.25, 0.125, 0.0625 ng of genomic DNA isolated from C2C12 cells, which have wild type dystrophin gene. PCR conditions were: 1 repetition at 50°C for 2 minutes, 1 repetition at 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Each experimental protocol was run in triplicate.
Co-transfection ssODNs and Msh2 RNAi
Purified myoblasts were plated in 6-well plates at a density of 1.5 × 105 cells/well with F10/DMEM growth medium 24 hours before transfection. Cells were co-transfected 0.75 mg of mdx47NT and siRNA for Msh2 knockdown (0–100 nM) using jetPEI. Twenty four hours after transfection, genomic DNA was isolated, and TaqMan® probe-based real time PCR was performed to quantify wild type dystrophin as described above ("Oligo dose experiment")
Purified myoblasts were plated in 6-well plates at a density of 1.5 × 105 cells/well with F10/DMEM growth medium 24 hours before transfection. Cells were co-transfected 0.75 mg of mdx47NT and siRNA for Msh2 knockdown (0–100 nM) using jetPEI. Total RNA was isolated 6 hours after transfection. Two hundred nanograms of RNA was subjected to reverse transcription to generate cDNA using the first strand synthesis kit (Invitrogen). Five microliters was added to a 20 ml mixture of 2× Sybergreen master mix (Applied Biosystems, Foster City, CA) with 2.5 mM of each primer: musMSH2/257F 5' agaccctgcagagtgttgtgctta 3'; musMSH2/1010R 5' tgagagccagtggtgtcttcaaca 3' or mouse GAPDH4FwQ 5' tgtctcctgcgacttcaacagc 3'; GAPDH5RevQ 5' atgtaggccatgaggtccacca 3'. Samples were added to each well of a 96 well plate in duplicate for each primer set. QPCR was run for 1 repetition at 50°C for 2 minutes, 1 repetition at 95°C for 10 minutes, 40 repetitions at 95°C for 15 seconds and 40 repetitions at 59°C for 1 minute with data collection at step 1 of stage 3. Upon completion of PCR, the threshold and baseline values were set to measure Ct values at the beginning of the exponential amplification. Ct values from 3 experiments were averaged per sample.
Duchenne Muscular Dystrophy.
We would like to thank Dr. J. Tremblay's lab for providing the enriched myoblasts and for critical reading of the manuscript (Unité de recherche en Génétique Humaine, Centre de Recherche du Centre Hospitalier de Université Laval, CHUL du CHUQ, Ste-Foy, QC, Canada). This project was supported by NIH R21 NS053608.
- Rando TA: The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies. Muscle Nerve. 2001, 24: 1575-1594. 10.1002/mus.1192View ArticlePubMedGoogle Scholar
- Tutdibi O, Brinkmeier H, Rudel R, Fohr KJ: Increased calcium entry into dystrophin-deficient muscle fibres of MDX and ADR-MDX mice is reduced by ion channel blockers. J Physiol. 1999, 515 (Pt 3): 859-868. 10.1111/j.1469-7793.1999.859ab.xPubMed CentralView ArticlePubMedGoogle Scholar
- Bertorini TE, Bhattacharya SK, Palmieri GM, Chesney CM, Pifer D, Baker B: Muscle calcium and magnesium content in Duchenne muscular dystrophy. Neurology. 1982, 32: 1088-1092.View ArticlePubMedGoogle Scholar
- Strober JB: Therapeutics in duchenne muscular dystrophy. NeuroRx. 2006, 3: 225-234. 10.1016/j.nurx.2006.01.005PubMed CentralView ArticlePubMedGoogle Scholar
- Harper SQ, Hauser MA, DelloRusso C, Duan D, Crawford RW, Phelps SF, et al: Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Nat Med. 2002, 8: 253-261. 10.1038/nm0302-253View ArticlePubMedGoogle Scholar
- Tinsley JM, Potter AC, Phelps SR, Fisher R, Trickett JI, Davies KE: Amelioration of the dystrophic phenotype of mdx mice using a truncated utrophin transgene. Nature. 1996, 384: 349-353. 10.1038/384349a0View ArticlePubMedGoogle Scholar
- Deconinck N, Tinsley J, De Backer F, Fisher R, Kahn D, Phelps S, et al: Expression of truncated utrophin leads to major functional improvements in dystrophin-deficient muscles of mice. Nat Med. 1997, 3: 1216-1221. 10.1038/nm1197-1216View ArticlePubMedGoogle Scholar
- Wang Z, Allen JM, Riddell SR, Gregorevic P, Storb R, Tapscott SJ, et al: Immunity to adeno-associated virus-mediated gene transfer in a random-bred canine model of Duchenne muscular dystrophy. Hum Gene Ther. 2007, 18: 18-26. 10.1089/hum.2006.093View ArticlePubMedGoogle Scholar
- Wang B, Li J, Fu FH, Xiao X: Systemic human minidystrophin gene transfer improves functions and life span of dystrophin and dystrophin/utrophin-deficient mice. J Orthop Res. 2008Google Scholar
- Bartlett RJ, Stockinger S, Denis MM, Bartlett WT, Inverardi L, Le TT, et al: In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide. Nat Biotechnol. 2000, 18: 615-622. 10.1038/76448View ArticlePubMedGoogle Scholar
- Rando TA, Disatnik MH, Zhou LZ: Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides. Proc Natl Acad Sci USA. 2000, 97: 5363-5368. 10.1073/pnas.97.10.5363PubMed CentralView ArticlePubMedGoogle Scholar
- Bertoni C, Rando TA: Dystrophin Gene Repair in mdx Muscle Precursor Cells In Vitro and In Vivo Mediated by RNA-DNA Chimeric Oligonucleotides. Hum Gene Ther. 2002, 13: 707-718. 10.1089/104303402317322276View ArticlePubMedGoogle Scholar
- Bertoni C, Lau C, Rando TA: Restoration of dystrophin expression in mdx muscle cells by chimeraplast-mediated exon skipping. Hum Mol Genet. 2003, 12: 1087-1099. 10.1093/hmg/ddg133View ArticlePubMedGoogle Scholar
- Pramono ZA, Takeshima Y, Alimsardjono H, Ishii A, Takeda S, Matsuo M: Induction of exon skipping of the dystrophin transcript in lymphoblastoid cells by transfecting an antisense oligodeoxynucleotide complementary to an exon recognition sequence. Biochem Biophys Res Commun. 1996, 226: 445-449. 10.1006/bbrc.1996.1375View ArticlePubMedGoogle Scholar
- Wilton SD, Lloyd F, Carville K, Fletcher S, Honeyman K, Agrawal S, et al: Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides. Neuromuscul Disord. 1999, 9: 330-338. 10.1016/S0960-8966(99)00010-3View ArticlePubMedGoogle Scholar
- Kole R, Sazani P: Antisense effects in the cell nucleus: modification of splicing. Curr Opin Mol Ther. 2001, 3: 229-234.PubMedGoogle Scholar
- Dunckley MG, Manoharan M, Villiet P, Eperon IC, Dickson G: Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides. Hum Mol Genet. 1998, 7: 1083-1090. 10.1093/hmg/7.7.1083View ArticlePubMedGoogle Scholar
- Mann CJ, Honeyman K, Cheng AJ, Ly T, Lloyd F, Fletcher S, et al: Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc Natl Acad Sci USA. 2001, 98: 42-47. 10.1073/pnas.011408598PubMed CentralView ArticlePubMedGoogle Scholar
- van Deutekom JC, Bremmer-Bout M, Janson AA, Ginjaar IB, Baas F, den Dunnen JT, et al: Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells. Hum Mol Genet. 2001, 10: 1547-1554. 10.1093/hmg/10.15.1547View ArticlePubMedGoogle Scholar
- Aartsma-Rus A, Bremmer-Bout M, Janson AA, den Dunnen JT, van Ommen GJ, van Deutekom JC: Targeted exon skipping as a potential gene correction therapy for Duchenne muscular dystrophy. Neuromuscul Disord. 2002, 12 (Suppl 1): S71-S77. 10.1016/S0960-8966(02)00086-XView ArticlePubMedGoogle Scholar
- Aartsma-Rus A, Janson AA, Kaman WE, Bremmer-Bout M, den Dunnen JT, Baas F, et al: Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum Mol Genet. 2003, 12: 907-914. 10.1093/hmg/ddg100View ArticlePubMedGoogle Scholar
- Vacek M, Sazani P, Kole R: Antisense-mediated redirection of mRNA splicing. Cell Mol Life Sci. 2003, 60: 825-833.PubMedGoogle Scholar
- Morgan JE, Partridge TA: Muscle satellite cells. Int J Biochem Cell Biol. 2003, 35: 1151-1156. 10.1016/S1357-2725(03)00042-6View ArticlePubMedGoogle Scholar
- Hawke TJ, Garry DJ: Myogenic satellite cells: physiology to molecular biology. J Appl Physiol. 2001, 91: 534-551.PubMedGoogle Scholar
- Bertoni C, Morris GE, Rando TA: Strand bias in oligonucleotide-mediated dystrophin gene editing. Hum Mol Genet. 2005, 14: 221-233. 10.1093/hmg/ddi020View ArticlePubMedGoogle Scholar
- Maguire KK, Kmiec EB: Multiple roles for MSH2 in the repair of a deletion mutation directed by modified single-stranded oligonucleotides. Gene. 2007, 386: 107-114. 10.1016/j.gene.2006.08.014PubMed CentralView ArticlePubMedGoogle Scholar
- Qu Z, Huard J: Matching host muscle and donor myoblasts for myosin heavy chain improves myoblast transfer therapy. Gene Ther. 2000, 7: 428-437. 10.1038/sj.gt.3301103View ArticlePubMedGoogle Scholar
- Rando TA, Blau HM: Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol. 1994, 125: 1275-1287. 10.1083/jcb.125.6.1275View ArticlePubMedGoogle Scholar
- Im WB, Phelps SF, Copen EH, Adams EG, Slightom JL, Chamberlain JS: Differential expression of dystrophin isoforms in strains of mdx mice with different mutations. Hum Mol Genet. 1996, 5: 1149-1153. 10.1093/hmg/5.8.1149View ArticlePubMedGoogle Scholar
- Aarts M, Dekker M, de Vries S, van der WA, Te RH: Generation of a mouse mutant by oligonucleotide-mediated gene modification in ES cells. Nucleic Acids Res. 2006, 34: e147- 10.1093/nar/gkl896PubMed CentralView ArticlePubMedGoogle Scholar
- Datta A, Adjiri A, New L, Crouse GF, Jinks RS: Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae. Mol Cell Biol. 1996, 16: 1085-1093.PubMed CentralView ArticlePubMedGoogle Scholar
- Selva EM, New L, Crouse GF, Lahue RS: Mismatch correction acts as a barrier to homeologous recombination in Saccharomyces cerevisiae. Genetics. 1995, 139: 1175-1188.PubMed CentralPubMedGoogle Scholar
- Hough SR, Wiederholt KA, Burrier AC, Woolf TM, Taylor MF: Why RNAi makes sense. Nat Biotechnol. 2003, 21: 731-732. 10.1038/nbt0703-731bView ArticlePubMedGoogle Scholar
- Liu L, Rice MC, Drury M, Cheng S, Gamper H, Kmiec EB: Strand bias in targeted gene repair is influenced by transcriptional activity. Mol Cell Biol. 2002, 22: 3852-3863. 10.1128/MCB.22.11.3852-3863.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Brachman EE, Kmiec EB: DNA replication and transcription direct a DNA strand bias in the process of targeted gene repair in mammalian cells. J Cell Sci. 2004, 117: 3867-3874. 10.1242/jcs.01250View ArticlePubMedGoogle Scholar
- Ferrara L, Kmiec EB: Targeted gene repair activates Chk1 and Chk2 and stalls replication in corrected cells. DNA Repair (Amst). 2006, 5: 422-431. 10.1016/j.dnarep.2005.11.009View ArticleGoogle Scholar
- Kinoshita I, Vilquin JT, Asselin I, Chamberlain J, Tremblay JP: Transplantation of myoblasts from a transgenic mouse overexpressing dystrophin prduced only a relatively small increase of dystrophin-positive membrane. Muscle Nerve. 1998, 21: 91-103. 10.1002/(SICI)1097-4598(199801)21:1<91::AID-MUS12>3.0.CO;2-3View ArticlePubMedGoogle Scholar
- Hu Y, Parekh-Olmedo H, Drury M, Skogen M, Kmiec EB: Reaction parameters of targeted gene repair in Mammalian cells. Mol Biotechnol. 2005, 29: 197-210. 10.1385/MB:29:3:197View ArticlePubMedGoogle Scholar
- Murphy BR, Moayedpardazi HS, Gewirtz AM, Diamond SL, Pierce EA: Delivery and mechanistic considerations for the production of knock-in mice by single-stranded oligonucleotide gene targeting. Gene Ther. 2007, 14: 304-315. 10.1038/sj.gt.3302866View ArticlePubMedGoogle Scholar
- Pierce EA, Liu Q, Igoucheva O, Omarrudin R, Ma H, Diamond SL, et al: Oligonucleotide-directed single-base DNA alterations in mouse embryonic stem cells. Gene Ther. 2003, 10: 24-33. 10.1038/sj.gt.3301857View ArticlePubMedGoogle Scholar
- Dekker M, Brouwers C, Te RH: Targeted gene modification in mismatch-repair-deficient embryonic stem cells by single-stranded DNA oligonucleotides. Nucleic Acids Res. 2003, 31: E27- 10.1093/nar/gng027PubMed CentralView ArticlePubMedGoogle Scholar
- Almofti MR, Harashima H, Shinohara Y, Almofti A, Li W, Kiwada H: Lipoplex size determines lipofection efficiency with or without serum. Mol Membr Biol. 2003, 20: 35-43. 10.1080/09687680210035104View ArticlePubMedGoogle Scholar
- Yamamoto T, Moerschell RP, Wakem LP, Komar-Panicucci S, Sherman F: Strand-specificity in the transformation of yeast with synthetic oligonucleotides. Genetics. 1992, 131: 811-819.PubMed CentralPubMedGoogle Scholar
- Morozov V, Wawrousek EF: Single-strand DNA-mediated targeted mutagenesis of genomic DNA in early mouse embryos is stimulated by Rad51/54 and by Ku70/86 inhibition. Gene Ther. 2008, 15: 468-472. 10.1038/sj.gt.3303088View ArticlePubMedGoogle Scholar