Human Molecular Genetics, 2002, Vol. 11, No. 19 2201-2206
© 2002 Oxford University Press
Validation in mesenchymal progenitor cells of a mutation-independent ex vivo approach to gene therapy for osteogenesis imperfecta
1Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin 2, Ireland and 2Programa de Terapias GÈnicas y Celulares, INTA, Universidad de Chile, Santiago, Chile
Received March 1, 2002; Revised July 3, 2002; Accepted July 7, 2002
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Over 100 dominant-negative mutations within the COL1A1 gene have been identified in osteogenesis imperfecta (OI). In terms of human therapeutics, targeting each of these mutations independently is unlikely to be feasible. Here we show that the hammerhead ribozyme Rzpol1a1, targeting a common polymorphism within transcripts from the COL1A1 gene, downregulates COL1A1 transcript in human mesenchymal progenitor cells at a ribozyme to transcript ratio of only 1:1. Downregulation was confirmed at the protein level. Transducing stem cells with Rzpol1A1 ex vivo followed by autologous transplantation could provide a gene therapy for a large proportion of OI patients with gain-of-function mutations using a single therapeutic.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Osteogenesis imperfecta (OI) is a debilitating disorder resulting in the formation of brittle bones. Types II, III and IV generally arise from gain-of-function mutations in either of the two type I collagen genes, COL1A1 and COL1A2, and represent serious, sometimes lethal, disorders. Type I OI, however, usually results from null mutations in the COL1A1 gene and is phenotypically milder (1–3). In this study we investigated a gene therapy for COL1A1-associated dominant-negative forms of OI in human mesenchymal progenitor cells (MPCs) using a hammerhead ribozyme. Notably, ribozymes have been shown to down-regulate COL1A1 transcripts specifically at mutation sites both in vitro (4) and in cellulo (5,6). In addition, mutation-independent ribozymes, targeting the COL1A1 and COL1A2 transcript in vitro, have been described (7,8).
The design of gene therapies for dominant hereditary diseases is greatly complicated by genetic heterogeneity. Gene therapies for dominant-negative diseases, such as many forms of OI, will probably require suppression of a mutant allele while still maintaining normal expression of a wild-type allele. Since over 100 dominant-negative mutations in the COL1A1 gene alone have been identified, the development of mutation-independent methods of suppressing mutant alleles (9) is imperative (for known mutations see www.le.ac.uk/ge/collagen/COL1A1.html). In this study, we examine the ability of a hammerhead ribozyme, Rzpol1a1, targeting a common C/T polymorphism at position 3210 (GenBank accession no. K01228) in the 3'UTR of the COL1A1 transcript (7,8,10), to downregulate COL1A1 in human MPCs. Rzpol1a1 can cleave the T allele of the polymorphism, but not the C allele in vitro (7,8). Since over 40% of Caucasian populations in the USA and Europe are heterozygous for the polymorphism (3,8), 20% of COL1A1-associated OI patients will harbour a mutation on the cleavable T allele and may therefore benefit from the effects of Rzpol1a1. Interestingly, the homozygous T/T population, representing an additional 40% of OI patients, may also be treated with Rzpol1a1. Since Rzpol1a1 would target both COL1A1 alleles, the therapy would need to include a replacement COL1A1 gene, altered at the ribozyme target site, such that it escapes ribozyme suppression. We cloned Rzpol1a1 into two different positions of a Moloney murine leukaemia viral vector (Mo-MLV), RNL-Pol (Fig. 1A), which we used to transfect human MPCs, homozygous for the T allele of COL1A1. Since RNL-Pol contains three different promoters, the 3'-LTR, Rous sarcoma virus (RSV) and cytomegalovirus (CMV) promoters, the virus should express three different RNA populations (Fig. 1B), enhancing ribozyme numbers and maximizing the chance of ribozyme activity.
|
Human MPCs were deemed to constitute a suitable system for testing Rzpol1a1 activity, since these cells can differentiate in vivo into osteoblasts, the cells involved in OI (11–13). It is notable that donor human MPCs have been administered to five children with severe OI. Engraftment was shown in three children, increasing their bone mineral content by between 44% and 77% of baseline values 3 months after treatment (14,15). Engrafted MPCs have been shown to constitute between 1% and 5% of bone marrow cells (14–19). While this demonstrates the administration of MPCs to patients and the cells' survival, it is, however, necessary to administer immunosuppressant drugs to patients treated in this manner, since acute and chronic graft versus host disease (GVHD) is a risk associated with bone marrow transplantation (15). In an alternative approach, utilizing autologous MPCs from OI patients as gene delivery vehicles in order to avoid the risk of GVHD, we have tested the mutation-independent hammerhead ribozyme, Rzpol1a1, in human MPCs. It is perhaps notable that Rzpol1a1 may also be delivered by other means. Regardless of the method of gene delivery, the functionality of any therapeutic drug in its target cell type must be investigated first. Thus, this study describes the functionality of a novel mutation-independent ribozyme, Rzpol1a1, targeting an intragenic polymorphism in COL1A1, in human MPCs, the target stem cell type for OI therapies.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
COL1A1 transcript downregulation in MPCs expressing Rzpol1a1
Having identified an MPC sample homozygous for the T allele of the COL1A1 gene, we stably transduced these cells with retroviral vector RNL-Pol (Fig. 1). Two MPC samples, MPC-Pol1 and MPC-Pol2, were thus generated. RNL-Pol carries the mutation-independent hammerhead ribozyme Rzpol1a1, which targets the T allele of COL1A1 specifically. COL1A1 transcript levels were assessed in MPC-Pol1, MPC-Pol2 and untransduced MPCs using real-time RT–PCR standardized with COL1A2 (the natural associate of COL1A1) and the housekeeping genes GAPDH and ß-actin. Downregulation of the COL1A1 transcript was confirmed 36 times in both MPC-Pol1 (Table 1) and MPC-Pol2 (Table 2). Mean levels of downregulation standardized with COL1A2, GAPDH and ß-actin of 40.31% (34.97%, 44.41% and 41.55%) and 47.08% (36.44%, 54.67% and 50.13%) were achieved in MPC-Pol1 and MPC-Pol2 respectively. Notably, differences in levels of COL1A1 suppression observed when RT–PCR reactions were standardized with COL1A2, GAPDH or ß-actin as internal controls were not significant in either MPC-Pol1 or MPC-Pol2 (P=0.72 and P=0.090 respectively).
|
|
Retroviral gene expression levels in MPC-Pol1 and MPC-Pol2
We compared levels of retroviral gene expression to COL1A1 expression levels in MPC-Pol1 and MPC-Pol2 in order to establish the level of Rzpol1a1 contributing to the observed downregulation of COL1A1. Expression levels were compared in terms of absolute copy number using real-time RT–PCR. Since RNL-Pol expresses three populations of RNA (Fig. 1B), two RT–PCR reactions were carried out. The first RT–PCR reaction amplified all three RNA populations arising from RNL-Pol. The second RT–PCR reaction amplified only the two larger RNA molecules from RNL-Pol. Results (Table 3) indicate that in both MPC-Pol1 and MPC-Pol2, a ratio of retroviral RNA to COL1A1 RNA of less than 1:1 is sufficient to achieve a downregulation of COL1A1 transcript of over 40% in human MPCs. In addition, these data indicate that in human MPCs the CMV promoter is estimated to be over five times stronger than the 5'-LTR and RSV promoters together.
|
Type I collagen protein downregulation in MPC-Pol1 and MPC-Pol2
To determine whether suppression of COL1A1 transcript is reflected at the protein level, we performed immunocytochemistry on untransduced MPCs, and MPC-Pol1 and MPC-Pol2 cells using a fluorescence isothiocyanate (FITC)-labelled anti-human type I collagen antibody. We used fibronectin as a control. Qualitative assessment after 4 and 7 days of cell growth showed reproducibly a marked downregulation of de novo type I collagen in MPC-Pol1 and MPC-Pol2 in terms of intensity of fluorescence, while fibronectin levels appeared to be unchanged. A representation of these data is presented in Figure 2.
|
Using ELISA as an alternative, quantifiable approach to monitoring protein downregulation, we assayed the amount of de novo type I collagen formed and excreted into the culture medium over a 24 h time period by untransduced MPCs, MPC-Pol1 and MPC-Pol2 cells. The relative amounts of de novo type I collagen found in media harvested from untransduced MPCs, MPC-Pol1 and MPC-Pol2 cells were 58.56±6.46 ng/ml, 36.67±4.68 ng/ml and 26.62±2.26 ng/ml respectively, indicating that the reduction in type I collagen levels found in MPC-Pol1 and MPC-Pol2 was 37.4% and 54.5%.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Rzpol1a1 is, to our knowledge, the first mutation-independent ribozyme to be active in cells and the first gene suppression agent that uses an single nucleotide polymorphism (SNP) to direct a therapy to a mutant allele in a mutation-independent manner. Polymorphisms have been invaluable in the generation of high-resolution genome maps and may have multiple future uses in the design of novel therapeutics. The current study provides a prototype for such a use.
We do not know what levels of mutant COL1A1 downregulation will be required to be of therapeutic benefit to OI patients. However, the quantification of relative levels of ribozyme to target transcript, suggests that even 1:1 ratios of Rzpol1a1 to COL1A1 transcript result in 40–50% suppression. This is encouraging, since the ratio of ribozyme to target may be augmented, resulting in an increase in levels of suppression. Notably, the suppression levels seen at the RNA level are almost exactly mirrored at the protein level as seen by ELISA and confirmed by immunocytochemistry. While measured levels of COL1A1 suppression varied between experiments, downregulation of RNA was significant and observed in all 72 experiments (Tables 1 and 2). Because expression levels of housekeeping genes are sometimes influenced by external factors (20), COL1A1 levels were compared to three very different genes, COL1A2, GAPDH and ß-actin. While measured levels of downregulation did vary depending on the internal control gene used, this variance was not significant. The variance is therefore likely to be a due to the resolution of real-time RT–PCR. Thus, we believe that suppression of COL1A1 is indeed due to Rzpol1a1 and not for example the retrovirus. Notably, real-time RT–PCR is the most accurate method of RNA quantification available at this time (http://dorakmt.tripod.com/genetics/realtime.html). The efficient downregulation of COL1A1 in MPCs and the use of a recombinant retrovirus to introduce multiple copies of the suppression agent, Rzpol1a1, represent additional novel aspects of the study. This is of particular interest given that the same stem cell type has previously been used in human clinical trials in OI patients. Results showing that the COL1A1 and CMV promoters are similar in strength in human MPCs highlight the necessity of using an extremely strong promoter to drive any gene therapeutic targeting COL1A1.
Hence, since Rzpol1a1 has been shown to promote significant suppression of COL1A1 at both the RNA and protein levels, we suggest that Rzpol1a1 may benefit over 20% (50% of the C/T 3210 COL1A1 heterozygous OI population) of OI patients with gain-of-function mutations in the COL1A1 gene. Interestingly, however, it may also be possible to treat homozygous COL1A1 3210 T-allele OI patients with Rzpol1a1 using a combined suppression and replacement strategy—representing an additional 40% of OI patients with COL1A1 mutations. In this situation, a replacement COL1A1 gene must be introduced concurrently with RNL-Pol and should be of the uncleavable 3210 C-allele type. In addition, the COL1A1 replacement gene may be altered at various sites surrounding the 3210 C polymorphism in the 3'-UTR, thus protecting replacement COL1A1 transcripts from ribozyme cleavage and suppression by the antisense arms of the ribozyme, without altering the collagen protein produced. Consequently, a single ribozyme, Rzpol1a1, may potentially be of therapeutic value to both COL1A1 3210C/T and 3210T/T OI patients, representing over 60% of COL1A1-linked OI cases, clearly demonstrating the power of polymorphism to overcome mutational heterogeneity in diseases such as OI. Despite significant downregulation at the RNA and protein levels of the target COL1A1 in human MPCs, it is worth noting that the degree of downregulation of mutant protein that would be required to provide therapeutic benefit to OI patients remains to be established.
In summary, this study amalgamates a novel aspect of the human genome (polymorphism) with suppression technologies (ribozymes), stem cell technologies (MPCs) and ex vivo delivery using retroviral vectors to overcome mutational heterogeneity, one of the most significant hurdles in the development of therapies for OI. It is of note that the genetic heterogeneity inherent in OI is mirrored in many other dominant disorders where hundreds of different mutations can give rise to clinically similar phenotypes. The immense number of SNPs now characterized in the human genome represents a powerful tool with which to attempt to overcome this heterogeneity when developing therapies, as has been clearly demonstrated in the current study.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
MPC isolation and culture
Heparinized human bone marrow cells, obtained from leftover materials of bone marrow transplantations, were used with informed consent. MPCs were isolated and cultured (21,22).
Retroviral construct and viral transfection of MPCs
Rzpol1a1 (8) was cloned into the SalI and BamHI sites of retroviral plasmid pLRNL. In addition, Rzpol1a1 driven by a CMV promoter was cloned into the ClaI site of pLRNL (23). An MLV virus, RNL-Pol, was generated with this construct (Fig. 1A) by the UCSD Program in Human Gene Therapy. MPCs were seeded at 2000 cells/cm2 and transfected at a multiplicity of infection (MOI)=5 in 83 µl/cm2 of 
-MEM, 20% fetal bovine serum (GibcoBRL, Paisley, UK; Cat. no. 10270–106) and 20 µg/ml of polybrene. Selection with 400 µg/ml G418 was started after 48 h. RNA was isolated using TriZol (GibcoBRL; Cat. no. 15596–018) and standard procedures 14 days later.
Cloning and in vitro transcription
Part of the COL1A1 coding sequence and 3'-UTR, spanning nucleotides 2977–3347 (GenBank accession no. K01228), was cloned into the HindIII and XbaI sites of pcDNA3 (Invitrogen, Groningen, The Netherlands; no longer available). In addition, a part of the neomycin gene, present in RNL-Pol (Fig. 1A), was cloned into the same sites of pcDNA3 using PCR and primers NEO F and NEO R (Table 4). Also, a section of the MLV envelope gene, 24 bp upsteam of the 3'-LTR in RNL-Pol, was cloned into the HindIII and XbaI sites of pcDNA3 using PCR and primers pLRNL F and pLRNL R (Table 4). The COL1A1, neo and env clones were maxiprepped using the HiSpeed Plasmid Maxi Kit (Qiagen, Crawley, UK; Cat. no. 12663) and linearized with BbsI. Digested clones were cleaned extensively with phenol, phenol–chloroform, chloroform and ethanol and used to express COL1A1, neo and env RNA using the T7 promoter present in pcDNA3, the Ribomax kit (Promega, Madison, Wisconsin, USA; Cat. no. P1300) and standard procedures. RNAs were treated with DNase for 20 min at 37°C. DNase was heat-inactivated for 5 min at 80°C. Copy numbers of RNA products were calculated from OD260 readings, performed in triplicate.
|
Real-time RT–PCR
COL1A1 downregulation experiments were performed four times in triplicate using real-time RT–PCR and a LightCycler (Roche, Lewes, UK). In addition, COL1A1 transcript levels were standardized using COL1A2, GAPDH and ß-actin as internal controls. Thus, in both MPC-Pol1 and MPC-Pol2, COL1A1 levels were quantified 36 times. RT–PCR reactions were carried out using the QuantiTect SYBR Green 1-step RT–PCR kit (Qiagen, UK; Cat. no. 204243), 57°C annealing temperatures, 5 s elongation times for COL1A1, COL1A2 and GAPDH, 10 s elongation times for ß-actin and the manufacturer's protocols. PCRs were carried out for between 28 and 35 cycles. RNA products expressed in vitro (see above) were used to generate standard curves of absolute copy numbers on a LightCycler, making the measurement of COL1A1 and retroviral transcript copy number in MPC-Pol1 and MPC-Pol2 possible. Thus the ratios of retroviral RNA to COL1A1 RNA were determined six times each in MPC-Pol1 and MPC-Pol2. All primers (Table 4) were HPLC-purified.
Immunocytochemistry
MPC-Pol1 and MPC-Pol2 cells were seeded at 2000 cells/cm2, grown for 4 or 7 days, fixed with 50% acetone and 50% methanol, and stained for 30 min at room temperature. Antibodies were anti-human type I collagen (BioDesign, Saco, Maine, USA; Cat. no. MIA340M) and monoclonal anti-human fibronectin (Sigma, Dublin, Ireland; Cat. no. F0916). The secondary antibody was anti-mouse IgG FITC conjugate (Sigma; Cat. no. F2012). Ninety-three fields of these cells were then photographed and assessed by 10 individuals in a blind test (data not shown).
ELISA assays
Two samples of untransduced MPCs, and MPC-Pol1 and MPC-Pol2 cells, were seeded at 2000 cells/cm2 and grown. Medium was removed and cells were washed three times with phosphate buffered saline (PBS). One millilitre of fresh medium, which did not contain fetal calf serum or antibiotics, was placed on cells, which were then grown for an additional 24 h. Medium was then removed and immediately stored at -80°C for use in ELISA assays. The numbers of MPC, MPC-Pol1 and MPC-Pol2 cells were then counted four times using a hemacytometer to determine the quantity of cells which had contributed to generating the de novo type I collagen excreted into the medium over 24 h. ELISAs on the six medium samples were performed in duplicate using the Metra CICP EIA kit (Metra Biosystems, San Diego, CA, USA; Cat. no. 8003) and the manufacturer's protocol. ELISA assays were analysed using MetraFIT, version 1.1.
Statistical analysis
Paired t-tests were performed using Data Desk 6.0 (Data Descriptions Inc., New York, New York, USA) to determine levels of COL1A1 downregulation. ANOVA tests were carried out to analyse whether results standardized with COL1A2, GAPDH and ß-actin were the same. Differences were considered significant at P<0.05. Standard deviations of ELISA results were calculated using Microsoft Excel 2002.
|
ACKNOWLEDGEMENTS |
|---|
We thank Irene Black for her tremendous help with the statistical analyses. We also thank the Children's Brittle Bone Foundation and the Health Research Board Ireland for their generous financial support and encouragement. We thank UNESCO/ISCU/TWAS and Fondecyt for supporting the collaboration between the laboratories in Dublin and Chile.
|
FOOTNOTES |
|---|
* To whom correspondence should be addressed at: Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin 2, Ireland. Tel: +353 16082482; Fax: +353 16083848; Email: sophia{at}maths.tcd.ie
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Sillence, D.O. and Rimoin, D.L. (1978) Classification of osteogenesis imperfect. Lancet, 1, 1041–1042.[Medline]
2 Sillence, D.O., Senn, A. and Danks, D.M. (1979) Genetic heterogeneity in osteogenesis imperfecta. J. Med. Genet., 16, 101–116.[Abstract]
3 Willing, M.C., Pruchno, C.J., Atkinson, M. and Byers, P.H. (1992) Osteogenesis imperfecta type I is commonly due to a COL1A1 null allele of type I collagen. Am. J. Hum. Genet., 51, 508–515.[ISI][Medline]
4
Grassi, G., Forlino, A. and Marini, J.C. (1997) Cleavage of collagen RNA transcripts by hammerhead ribozymes in vitro is mutation-specific and shows competitive binding effects. Nucleic Acids Res., 25, 3451–3458.
5
Dawson, P.A. and Marini, J.C. (2000) Hammerhead ribozymes selectively suppress mutant type I collagen mRNA in osteogenesis imperfecta fibroblasts. Nucleic Acids Res., 28, 4013–4020.
6 Toudjarska, I., Kilpatrick, M.W., Niu, J., Wenstrup, R.J. and Tsipouras, P. (2001) Delivery of a hammerhead ribozyme specifically downregulates mutant type I collagen mRNA in a murine model of osteogenesis imperfecta. Antisense Nucleic Acid Drug Dev., 11, 341–346.[ISI][Medline]
7
Millington-Ward, S., O'Neill, B., Tuohy, G., Al-Jandal, N., Kiang, A.S., Kenna, P.F., Palfi, A., Hayden, P., Mansergh, F., Kennan, A. et al. (1997) Strategems in vitro for gene therapies directed to dominant mutations. Hum. Mol. Genet., 6, 1415–1426.
8 Millington-Ward, S., O'Neill, B., Kiang, A.S., Humphries, P., Kenna, P.F. and Farrar, G.J. (1999) A mutation-independent therapeutic strategem for osteogenesis imperfecta. Antisense Nucleic Acid Drug Dev., 9, 537–542.[ISI][Medline]
9
O'Neill, B., Millington-Ward, S., O'Reilly, M., Tuohy, G., Kiang, A.S., Kenna, P.F., Humphries, P. and Farrar, G.J. (2000) Ribozyme-based therapeutic approaches for autosomal dominant retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci., 41, 2863–2869.
10
Westerhausen, A., Leicht, W. and Heinz, F. (1990) A sequence polymorphism in the 3'-nontranslated region of the pro alpha 1 chain of type I procollagen. Nucleic Acids Res., 18, 4968.
11
Prockop, D.J. (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science, 276, 71–74.
12
Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S. and Marshak, D.R. (1999) Multilineage potential of adult human mesenchymal stem cells. Science, 284, 143–147.
13 Zvaifler, N.J., Marinova-Mutafchieva, L., Adams, G., Edwards, C.J., Moss, J., Burger, J.A. and Maini, R.N. (2000) Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res., 2, 477–488.[ISI][Medline]
14 Horwitz, E.M., Prockop, D.J., Fitzpatrick, L.A., Koo, W.W., Gordon, P.L., Neel, M., Sussman, M., Orchard, P., Marx, J.C., Pyeritz, R.E. and Brenner, M.K. (1999) Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat. Med., 5, 309–313.[ISI][Medline]
15
Horwitz, E.M., Prockop, D.J., Gordon, P.L., Koo, W.W., Fitzpatrick, L.A., Neel, M.D., McCarville, M.E., Orchard, P.J., Pyeritz, R.E. and Brenner, M.K. (2001) Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood, 97, 1227–1231.
16
Pereira, R.F., O'Hara, M.D., Laptev, A.V., Halford, K.W., Pollard, M.D., Class, R., Simon, D., Livezey, K. and Prockop, D.J. (1998) Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta. Proc. Natl Acad. Sci. USA, 95, 1142–1147.
17 Onyia, J.E., Clapp, D.W., Long, H. and Hock, J.M. (1998) Osteoprogenitor cells as targets for ex vivo gene transfer. J. Bone Miner. Res., 13, 20–30.[ISI][Medline]
18 Ding, L., Lu, S., Batchu, R., III, R.S. and Munshi, N. (1999) Bone marrow stromal cells as a vehicle for gene transfer. Gene Ther., 6, 1611–1616.[ISI][Medline]
19 Primorac, D., Rowe, D.W., Mottes, M., Barisic, I., Anticevic, D., Mirandola, S., Gomez Lira, M., Kalajzic, I., Kusec, V. and Glorieux, F.H. (2001) Osteogenesis imperfecta at the beginning of bone and joint decade. Croat. Med. J., 42, 393–415.[ISI][Medline]
20 Selvey, S., Thompson, E.W., Matthaei, K., Lea, R.A., Irving, M.G. and Griffiths, L.R. (2001) beta-Actin—an unsuitable internal control for RT–PCR. Mol. Cell Probes, 15, 307–311.[ISI][Medline]
21 Conget, P.A. and Minguell, J.J. (1999) Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J. Cell Physiol., 181, 67–73.[ISI][Medline]
22 Conget, P.A. and Minguell, J.J. (2000) Adenoviral-mediated gene transfer into ex vivo expanded human bone marrow mesenchymal progenitor cells. Exp. Hematol., 28, 382–390.[ISI][Medline]
23
Yee, J.K., Miyanohara, A., LaPorte, P., Bouic, K., Burns, J.C. and Friedmann, T. (1994) A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes. Proc. Natl Acad. Sci. USA, 91, 9564–9568.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Shivapurkar, V. Stastny, N. Okumura, L. Girard, Y. Xie, C. Prinsen, F. B. Thunnissen, I. I. Wistuba, B. Czerniak, E. Frenkel, et al. Cytoglobin, the Newest Member of the Globin Family, Functions as a Tumor Suppressor Gene Cancer Res., September 15, 2008; 68(18): 7448 - 7456. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. B. J. Glass, S. K. Fernbach, K. I. Norton, P. S. Choi, and T. P. Naidich The Infant Skull: A Vault of Information RadioGraphics, March 1, 2004; 24(2): 507 - 522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Abdelgany, M. Wood, and D. Beeson Allele-specific silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference Hum. Mol. Genet., October 16, 2003; 12(20): 2637 - 2644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J.A. Wood, B. Trulzsch, A. Abdelgany, and D. Beeson Therapeutic gene silencing in the nervous system Hum. Mol. Genet., October 15, 2003; 12(90002): R279 - 284. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||