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Nucleofection is highly efficient for transfecting genes into murine embryonic palatal mesenchymal cells in primary culture
Int. J. Oral Maxillofac. Surg. 2007; 36: 429–434 doi:10.1016/j.ijom.2006.12.008, available online at http://www.sciencedirect.com
Research Paper Congenital Craniofacial Anomalies
Nucleofection is highly efficient for transfecting genes into murine embryonic palatal mesenchymal cells in primary culture
W.-L. Xiao1,2, B. Shi1, Q. Zheng1, Y. Wang1, L. Huang1, S. Li1, Y. Lu1, M. Wu3 1 Department of Oral and Maxillofacial Surgery, West China College of Stomatology, Sichuan University, Chengdu, People’s Republic of China; 2Department of Oral and Maxillofacial Surgery, The Medical School Hospital of Qingdao University, Qingdao, People’s Republic of China; 3Department of Biochemistry & Molecular Biology, University of North Dakota, Grand Forks, ND, USA
W.-L. Xiao, B. Shi, Q. Zheng, Y. Wang, L. Huang, S. Li, Y. Lu, M. Wu: Nucleofection is highly efficient for transfecting genes into murine embryonic palatal mesenchymal cells in primary culture. Int. J. Oral Maxillofac. Surg. 2007; 36: 429–434. # 2007 Published by Elsevier Ltd on behalf of International Association of Oral and Maxillofacial Surgeons. Abstract. Non-syndromic cleft of the lip and/or palate is one of the most common birth defects in humans. Embryonic palatal mesenchymal (EPM) cells are an attractive source for investigating embryonic palatal development. In this study, we developed a highly efficient transfection method for murine EPM (MEPM) cells. MEPM cells were transfected with the plasmid pEGFP-N1 using two non-viral methods: nucleofection and lipofection. Nucleofection provided a much better rate of gene transfer than lipofection particularly in MEPM cells. The methylenetetrahydrofolate reductase (MTHFR) gene is an important candidate for involvement in the pathogenesis of this birth defect. The RNA interference plasmid of MTHFR was constructed and nucleofected into MEPM cells. Successful transfection resulted in a remarkable reduction in the expression of MTHFR. Taken together, the results indicate that nucleofection is highly efficient for MEPM cell transfection, and that this approach may be useful for investigating gene function in the process of palatogenesis.
The etiology of non-syndromic cleft of the lip and/or palate (NCLP) is thought to be multifactorial, including genetic and environmental factors21. Embryonic palatal mesenchymal (EPM) cells are derived from embryonic palate. Primary culture of murine and human EPM cells has been described14,17,32,33 and used extensively to 0901-5027/050429 + 06 $30.00/0
investigate the mechanism of the interaction between genetic and environmental factors in the process of palatogenesis8,34. RNA interference (RNAi) as a strategy for reducing RNA expression has been proven to be a powerful tool for reverse genetic analysis20. RNAi has also shown great promise in dissecting the pathogenic
Keywords: cleft palate; RNA interference; mouse embryo; palatal mesenchymal cells; MTHFR; transfection. Accepted for publication 11 December 2006
mechanisms of many organisms11. To obtain meaningful results, this technique requires significant knockdown of a target gene. Many different approaches have been used for transfecting exogenous DNAs into primary culture cells2,3,6, but transfection with EPM cells is rarely explored
# 2007 Published by Elsevier Ltd on behalf of International Association of Oral and Maxillofacial Surgeons.
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and no satisfactory methods are currently available. Lipofectin is commonly used for transfection of RNAi, but with low efficiency. Viral vectors and electroporation may be more efficient but for technical reasons their use has often been limited. Viral vectors introduce an exogenous DNA in the form of viral genes that can complicate the analysis of gene expression, and the vectors are sometimes inactivated through methylation. Electroporation conditions that elicit the highest gene expression may also yield more cell death24. The recent development of NucleofactorTM technology allows directed electroporation of an exogenous DNA to the nucleus, and this technology has been applied successfully in transfection of primary culture cells that are generally difficult to transfect18,35. Here is described a novel nucleofection medium and conditions for reproducible transient delivery of exogenous pEGFPN1 to murine EPM (MEPM) cells. Importantly, the method was used to show knockdown of an endogenous gene, methylenetetrahydrofolate reductase (MTHFR), using a specific RNAi plasmid. The novel transfection protocol may allow delineation of gene function in MEPM cells, which was previously not feasible due to inability to achieve high efficiency transfection in this cell type. Materials and methods Cell culture
C57BL/6J mice aged 8 weeks (Jackson Laboratory, Bar Harbor, MI, USA) were maintained at a temperature of 22 8C with an alternating light/dark cycle and were given access to food and filtered water. Mature male and female (8 weeks old) were mated overnight and the presence of a vaginal plug the following morning was taken as evidence of mating (gestation day 0, GD0). Palate shelves were dissected from GD13 embryos by using a surgical microscope, as described previously3. Removed palatal shelves were washed three times with Ca–Mg free 0.01 M phosphate-buffered saline (PBS), and incubated with dispase (Gibco BRL, Life Technologies, Grand Island, NY, USA) for 18 h at 4 8C. An operation was performed according to the method described previously28 to separate the palatal mesenchyme and epithelia. The purified palatal mesenchymes was incubated with 0.25% trypsin, 0.02% EDTA in Ca–Mg free PBS for 15 min at 37 8C to dissociate individual cells. The action of trypsin was inhibited
by 4 8C Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s Medium F12 (1:1) containing 10% fetal bovine serum (FBS). The cell number was then determined using a hemocytometer. Primary cultures of MEPM cells were initiated by seeding 5 105 cells/5 ml DMEM/F12 with 10% FBS into a 25-ml tissue-culture flask. Confluent MEPM cells were subcultured at a ratio of 1:3. Transfection of pEGFP-N1 by nucleofection and lipofection
The pEGFP-N1 plasmid was purchased from Clontech (Palo Alto, CA, USA) and used to appreciate the transfection efficiency. All experiments for transfection were repeated three times. The primary culture of MEPM cells was transfected using Amaxa nucleofectorTM technology (Amaxa, Koeln, Germany). Three nucleofection programs, A23, G16, and U30, were selected to test the transfection efficiency in MEPM cells. The cells (1 106) were resuspended in solution from nucleofector Kit R, also available as part of the Amaxa cell optimization kit, following the manufacturer’s instructions for cell transfection. The cell suspension was mixed with 5 mg purified pEGFP-N1 and nucleofected using nucleofection program A23, G16 or U30. After transfection, the cells were transferred to a six-well plate with 2 ml DMEM/F12 complete media. The percentage of GFP-positive MEPM cells was assessed at 48 h after transfection using a fluorescence microscope. For comparison, pEGFP-N1 was transfected to MEPM cells by lipofection. The plasmid was mixed with Lipofectamine 2000TM according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). Briefly, for each transfection, 10 ml Lipofectamine 2000 was diluted with 250 ml serum-free OptiMEM and kept at room temperature for 5 min, mixed with 250 ml serum-free Opti-MEM containing 4 mg pEGFP-N1 plasmid DNA, and the mixture was then left at room temperature for 20 min. Prior to transfection, cells in six-well plates were rinsed twice with serum-free Opti-MEM. The above DNA–Lipofectamine 2000 complex was added to the MEPM cells. After incubation at 37 8C for 12 h, the transfection medium was replaced with fresh DMEM/F12 complete medium. The percentage of GFPpositive MEPM cells was assessed at 48 h after transfection using a fluorescence microscope.
Transfection efficiency analysis by fluorescence microscopy
The transfected MEPM cells were viewed for fluorescent protein expression using a fluorescence microscope (Olympus IX50, Olympus, Japan) equipped with phase-contrast illumination, and photographs were captured directly using a digital camera. Nine areas were randomly captured for each well in order to obtain 200 cells for analysis. The total number of MEPM cells within the areas was determined using phase contrast. Green fluorescent protein (GFP)-positive MEPM cells were also counted in the same areas. This allowed calculation of the percentage of GFP-positive MEPM cells compared to the total number of cells in all nine areas as described previously19. The percentage of GFP-positive MEPM cells represents the transfection efficiency. Construction RNAi plasmid of MTHFR gene
Four small iRNA (siRNA) sequences were designed according to the cDNA of MTHFR in GenBank (GenBank accession No. AK030192)9. The sense sequences were: S-1 GGCCT ACCTC GAATT CTTC; S-2 GAGCT ACATC TACCG CACA; S-3 CCAGC CTGAT GAAGG AAGA; S-4 GGATG TAATT GAGCC CATC. The four siRNAs suppressed mRNA expression of the MTHFR gene using the LineSilence RNATM kit (Allele Biotechnology & Pharmaceuticals, San Diego, CA, USA) as described previously7. A semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay showed that the S-3 sequence was the most efficient at screening mRNA expression of the MTHFR gene. The shRNA (short-hairpin RNA) sequence was synthesized according to S-3 and cloned into psiRNA-Hh1neo_G2 (psiRNA-MTHFR) (InvivoGen, San Diego, CA, USA). The corresponding scrambled sequence of S-3 (in which all of the bases were randomly aligned) was analysed using BLAST to eliminate the possibility of anyone genes. The shRNA sequence was then synthesized according to the nonsense strand sequence of S-3, and cloned into psiRNA-Hh1neo_G2 (psiRNA-NMTHFR). Transfection of RNAi plasmids targeting the MTHFR gene to MEPM cells
MEPM cells were nucleofected with psiRNA-Hh1neo_G2, psiRNA-MTHFR or psiRNA-NMTHFR by nucleofection
Efficiency of nucleofection in transfecting genes program U30 using the solution from the nucleofector Kit R (Amaxa). After nucleofection, the cells were seeded in six-well plates to culture for 48 h before analysing transfection efficiency.
Quantitative real-time PCR
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Statistical analysis
Results
Data analysis was performed using the SPSS statistics software package. All the results were expressed as means SEM and n refers to the number of experiments. P < 0.05 was considered statistically significant.
The MEPM cells transfected by nucleofection and lipofection with pEGFP-N1 vector were analysed by fluorescence microscopy 48 h after transfection (Fig. 1). The number of GFP-expressing cells (92%) indicated very high levels of
Total RNA was extracted by Trizol reagent (Gibco BRL) according to the manufacturer’s instructions. RNA was reversely transcribed to cDNA using a kit (Promega, Madison, WI). Real-time PCR reactions were carried out using a real-time PCR kit (Takara, Japan). The levels of GAPDH mRNA were quantified as an internal control. The RT mixture was diluted to 100 ml with glass-distilled water. The PCR products were detected using a real-time system FTC 2000 (Funglyn, Toronto, Canada). PCR reactions were performed in triplicate for each sample and the mean value at which the PCR product crossed the threshold (Ct) was calculated. The ratio of MTHFR mRNA expression to that of the internal standard mRNA (GAPDH) was calculated using a method described previously23.
Western blotting
Cells were harvested at different time points for protein extraction with mammalian protein extraction reagent (M-PER; Pierce, Rockford, IL, USA). Equal amounts of total protein were electrophoresed in a 10% SDS–PAGE gel, and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA) in a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, Richmond, CA, USA) at 15 V for 30 min. The membrane was blocked for 2 h at room temperature with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TTBS) and incubated overnight with primary polyclonal antiMTHFR antibodies (Santa Cruz, CA, USA) (1:2000 dilution), monoclonal anti-b-actin antibody (Sigma-Aldrich, St Louis, MO, USA) (1:5000 dilution). The membrane was then probed with secondary horse radish peroxidase-conjugated anti-goat antibodies (1:5000 dilution in TTBS; Santa Cruz) for 1 h and developed with an ECL Detection kit (Amersham, Uppsala, Sweden) according to the manufacturer’s instructions. The protein was detected with an ECL system (Amersham) by chemoluminescence, and visualized on radiographic film. Protein expression was quantified with Gel-Pro Analyzer 3.1 software (Media Cybernetics, Silver Spring, MD, USA).
Fig. 1. Transfection efficiency of nucleofection and lipofection for MEPM cells using pGEFPN1 analyzed by fluorescence microscopy at 24 h after transfection. A, C, E, G show phase contrast and B, D, F, H show fluorescent pictures (magnification: 100). MEPM cells were nucleofected using program A23 (A and B), G16 (C and D) or U30 (E and F). MEPM cells (G and H) were transfected using Lipofectamine 2000. The highest efficiency of transfection was obtained with nucleofection program U30. The efficiency was respectively 60 and 42% using program G16 and A23. Efficiency using lipofection was only 15%.
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Fig. 2. Significant reduction in MTHFR mRNA expression by psiRNA-MTHFR transfection. Primary cultures of MEPM cells (1 106 cells) were transfected using program U30 and the solution from nucleofector Kit R with 4 mg highly purified plasmid DNA. psiRNA-MTHFR: RNAi vector; psiRNA-NMTHFR: RNAi nonsense strands vector; psiRNA: psiRNA-Hh1neo_G2 alone vector; control: blank control. Real-Time PCR results of MTHFR mRNA expression after MTHFR gene silencing for 48 h. MTHFR mRNA expression was suppressed significantly by psiRNA-MTHFR compared to control (n = 3,*P < 0.001).
fluorescence in the nucleofected cells using nucleofection program U30. The number of GFP-expressing cells was 60% and 42% using nucleofection programs G16 and A23, respectively. All three nucleofection programs for MEPM cells did not result in any visible toxic side effects. In contrast, lipofection resulted in fewer cells with GFP expression (15%). An RNAi plasmid targeting the endogenous MTHFR gene was nucleofected to MEPM cells for studying MTHFR genesilencing efficiency. The constructed RNAi plasmid was nucleofected to MEPM cells using the most efficient nucleofection method (nucleofection program U30 and the solution from nucleofector Kit R). Using real-time PCR, it was found that the mRNA level of MTHFR was reduced by 88% at 48 h (P < 0.001) compared to control (Fig. 2). Western
blotting analysis demonstrated a significant reduction (80%) of MTHFR protein expression in MEPM cells 48 h after nucleofection compared to control (P < 0.001, Fig. 3). Discussion
EPM cells derived from embryonic palate form the bone, muscular and adipose tissue of the palate26, and these cells could be identified by staining with vimentin, S100 and desmin32. Vimentin is used as a marker of mesenchymal tissue31, S-100 protein is used as a marker of neural tissue1 and desmin is a marker of muscle tissue25. EPM cells thus have the potential to differentiate into a variety of cells with mesenchymal, neural and myoblastoid characteristics. Three nucleofection programs were selected according to the
Fig. 3. Decrease in MTHFR protein expression by western blotting analysis. Transfection was performed as above described. MTHFR protein was about 74KD and the expression levels of MTHFR protein were suppressed significantly after MTHFR gene silencing for 48 h. An anti-bactin antibody was used as an internal control. Lanes a–d: (a) control, (b) psiRNA-NMTHFR, (c) psiRNA, (d) psiRNA-MTHFR. MTHFR protein of MTHFR gene silencing for 48 h was quantitated by densitometric analysis (n = 3). *P < 0.001, indicating significant inhibition compared to control.
above characteristics of EPM cells. The highest efficiency of transfection was obtained with the program U30 without significant cell toxicity. Although lipofection has been used to transfect plasmid to EPM cells for investigating the mechanism of palatogenesis14, the transfection efficiency was unknown. The transfection efficiency of lipofection in the present study was much lower than that of nucleofection. The etiology of NCLP is thought to be multifactorial, with both genetic and environmental factors playing a role. The MTHFR gene is located on chromosome 1q36 and is a key enzyme in folic acid metabolism13. C677T (alanine to valine) was the first common MTHFR variant identified to correlate with reduced enzyme activity and increased thermolability in both the heterozygous and homozygous states12. A second common MTHFR variant, A1298C (glutamate to alanine), has also been associated with decreased enzyme activity, although to a lesser extent than C677T29. Mothers carrying the MTHFR 677TT genotype who either did not use folic acid supplements periconceptionally or had a low dietary folate intake, or both, had an increased risk of delivering a CLP child by almost sixfold, three-fold and 10-fold, respectively30. No folate supplement use, low dietary folate intake and maternal MTHFR 1298CC genotype increased the risk of CLP offspring almost seven-fold30. Some epidemiological studies also indicated that the interaction between the MTHFR gene and folic acid was a key factor during palatogenesis. The present study confirmed that the RNAi plasmid of the MTHFR gene could be nucleofected efficiently into MEPM cells, and that the expression of MTHFR in MEPM cells was remarkably inhibited. The nucleofection protocol may be used to investigate the mechanism of folic acid supplements by preventing the teratogenic effect of MTHFR mutation in cultured MEPM cells. The nucleofection protocol may also be applied to analyse the pathogenesis of the other candidate genes in NCLP, a number of which (MSX1, PVRL1, TGFb, etc.) have been identified in recent years4,10. With regard to a functional gene, conventional antisense RNA and gene knockout are the all-purpose methods of reverse genetics5,22. Gene knockout could eliminate completely the activity of a target gene while not necessarily reflecting the specific function of the gene at a particular embryonic developmental stage. The antisense RNA technology is not sufficient to
Efficiency of nucleofection in transfecting genes suppress the expression of an endogenous gene and results in a transitional phenotype, misleading our interpretation of the function of a particular gene. The ability of RNAi to efficiently suppress target genes in mammals has been heralded as the most powerful reverse genetics approach in recent years. Up to now, the mechanism of palatogenesis has been studied from the cell to organ level16,27. To further understand the role of candidate gene variants in palatogenesis, RNAi may be a useful tool. By nucleofecting the RNAi plasmid of different candidate genes into MEPM cells, the inherent mechanism of the genes resulting in cleft palate could be elucidated. It will also be worthwhile to nucleofect RNAi plasmid of different candidate genes into cultured embryonic palatal shelves of animal models to investigate the mechanisms of palatal shelf confluence at the organ level. If these candidate genes function and the cross-talk between them is elucidated, it will be possible to prenatally diagnose NCLP and reduce its incidence by preventive treatment. Transfection of primary cells is challenging15. Transfection of EPM cells has rarely been studied, and without much success. Lipofectin is commonly used for transfection of RNAi, but with low efficiency. Viral vectors and electroporation may be more efficient but for technical reasons their use has often been limited. The newer method of nucleofection is simple and has proven to be efficient in transfecting various primary cells18,35. In the present study, it was shown that the nucleofection of MEPM cells provided a method for rapid and efficient transfection. The Amaxa transfection system, with nucleofection program U30 and the solution from nucleofector Kit R, was a very efficient way to transfect the MEPM cells. This method may be useful for delineating the function of candidate genes during palatogenesis by using RNAi technology. Acknowledgement. This work was supported by grants from the National Natural Science Foundation of China (30371552).
References 1. Anneren G, Esscher T, Larsson L, Olsen L, Pahlman S. S-100 protein and neuron-specific enolase in amniotic fluid as markers of abdominal wall and neural tube defects in the foetus. Prenat Diagn 1988: 8: 323–328.
2. Behr JP, Demeneix B, Loeffler JP, Perez-Mutul J. Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc Natl Acad Sci U S A 1989: 86: 6982– 6986. 3. Bierhuizen MF, Westerman Y, Visser TP, Wognum AW, Wagemaker G. Green fluorescent protein variants as markers of retroviral-mediated gene transfer in primary hematopoietic cells and cell lines. Biochem Biophys Res Commun 1997: 234: 371–375. 4. Carinci F, Pezzetti F, Scapoli L, Martinelli M, Avantaggiato A, Carinci P, Padula E, Baciliero U, Gombos F, Laino G, Rullo R, Cenzi R, Carls F, Tognon M. Recent developments in orofacial cleft genetics. J Craniofac Surg 2003: 14: 130–143. 5. Chen Z, Karaplis AC, Ackerman SL, Pogribny IP, Melnyk S, LussierCacan S, Chen MF, Pai A, John SW, Smith RS, Bottiglieri T, Bagley P, Selhub J, Rudnicki MA, James SJ, Rozen R. Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition. Hum Mol Genet 2001: 10: 433–443. 6. Chu G, Hayakawa H, Berg P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res 1987: 15: 1311–1326. 7. Csiszar A, Ungvari Z, Koller AG, Edwards J, Kaley G. Proinflammatory phenotype of coronary arteries promotes endothelial apoptosis in aging. Physiol Genomics 2004: 17: 21–30. 8. Dhulipala VC, Hanumegowda UM, Balasubramanian G, Reddy CS. Relevance of the palatal protein kinase A pathway to the pathogenesis of cleft palate by secalonic acid D in mice. Toxicol Appl Pharmacol 2004: 194: 270–279. 9. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21and 22-nucleotide RNAs. Genes Dev 2001: 15: 188–200. 10. Farrall M, Holder S. Familial recurrence-pattern analysis of cleft lip with or without cleft palate. Am J Hum Genet 1992: 50: 270–277. 11. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998: 391: 806–811. 12. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995: 10: 111–113. 13. Goyette P, Pai A, Milos R, Frosst P, Tran P, Chen Z, Chan M, Rozen R. Gene structure of human and mouse
14.
15.
16.
17.
18.
19.
20.
21. 22.
23.
24.
25.
26.
433
methylenetetrahydrofolate reductase (MTHFR). Mamm Genome 1998: 9: 652–656. Greene RM, Nugent P, Mukhopadhyay P, Warner DR, Pisano MM. Intracellular dynamics of Smad-mediated TGFbeta signaling. J Cell Physiol 2003: 197: 261–271. Gresch O, Engel FB, Nesic D, Tran TT, England HM, Hickman ES, Korner I, Gan L, Chen S, Castro-Obregon S, Hammermann R, Wolf J, Muller-Hartmann H, Nix M, Siebenkotten G, Kraus G, Lun K. New nonviral method for gene transfer into primary cells. Methods 2004: 33: 151–163. Han J, Xiao Y, Lin J, Li Y. PDGF-C Controls proliferation and is down-regulated by retinoic acid in mouse embryonic palatal mesenchymal cells. Birth Def Res B Dev Reprod Toxicol 2006: 77: 438– 444. Hanumegowda UM, Dhulipala VC, Reddy CS. Mechanism of secalonic acid D-induced inhibition of transcription factor binding to cyclic AMP response element in the developing murine palate. Toxicol Sci 2002: 70: 55–62. Lakshmipathy U, Pelacho B, Sudo K, Linehan JL, Coucouvanis E, Kaufman DS, Verfaillie CM. Efficient transfection of embryonic and adult stem cells. Stem Cells 2004: 22: 531–543. Leclere PG, Panjwani A, Docherty R, Berry M, Pizzey J, Tonge DA. Effective gene delivery to adult neurons by a modified form of electroporation. J Neurosci Methods 2005: 142: 137– 143. Morris JC, Wang Z, Drew ME, Englund PT. Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library. EMBO J 2002: 21: 4429–4438. Murray JC. Gene/environment causes of cleft lip and/or palate. Clin Genet 2002: 61: 248–256. O’Brodorich H. Fetal lung liquid secretion: insights using the tools of inhibitors and genetic knock-out experiments. Am J Respir Cell Mol Biol 2001: 25: 8–10. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002: 30: e36. Pinero J, Lopez-Baena M, Ortiz T, Cortes F. Apoptotic and necrotic cell death are both induced by electroporation in HL60 human promyeloid leukaemia cells. Apoptosis 1997: 2: 330–336. Schaart G, Viebahn C, Langmann W, Ramaekers F. Desmin and titin expression in early postimplantation mouse embryos. Development 1989: 107: 585– 596. Sperber GH, Machin GA. The enigma of cephalogenesis. Cleft Palate Craniofac J 1994: 31: 91–96.
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Xiao et al.
27. Sun D, Vanderburg CR, Odierna GS, Hay ED. TGFbeta3 promotes transformation of chicken palate medial edge epithelium to mesenchyme in vitro. Development 1998: 125: 95–105. 28. Tyler MS, Pratt RM. Effect of epidermal growth factor on secondary palatal epithelium in vitro: tissue isolation and recombination studies. J Embryol Exp Morphol 1980: 58: 93–106. 29. van der Put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes TK, van den Heuvel LP, Blom HJ. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet 1998: 62: 1044–1051. 30. van Rooij IA, Vermeij-Keers C, Kluijtmans LA, Ocke MC, Zielhuis GA, Goorhuis-Brouwer SM, van der Biezen JJ, Kuijpers-Jagtman AM, Steegers-Theunissen RP. Does the interaction between maternal folate
31.
32.
33.
34.
intake and the methylenetetrahydrofolate reducetase polymorphisms affect the risk of cleft lip with or without cleft palate? Am J Epidemiol 2003: 157: 583–591. von Koskull H, Virtanen I. Induction of cytokeratin expression in human mesenchymal cells. J Cell Physiol 1987: 133: 321–329. Yano H, Yoshimoto H, Ohtsuru A, Ito M, Yamashita S, Fujii T. Characterization of cultured rat embryonic palatal mesenchymal cells. Cleft Palate Craniofac J 1996: 33: 379–384. Yoneda T, Urade M, Sakuda M, Miyazaki T. Altered growth, differentiation, and responsiveness to epidermal growth factor of human embryonic mesenchymal cells of palate by persistent rubella virus infection. J Clin Invest 1986: 77: 1613– 1621. Yu Z, Xing Y. All-trans retinoic acid inhibited chondrogenesis of mouse embryonic palate mesenchymal cells by down-regulation of TGF-beta/Smad sig-
naling. Biochem Biophys Res Commun 2006: 340: 929–934. 35. Zernecke A, Erl W, Fraemohs L, Lietz M, Weber C. Suppression of endothelial adhesion molecule up-regulation with cyclopentenone prostaglandins is dissociated from IkappaB-alpha kinase inhibition and cell death induction. FASEB J 2003: 17: 1099–1101. Address: Bing Shi Department of Oral and Maxillofacial Surgery West China College of Stomatology of Sichuan University No. 14 Section 3 Ren Min Nan Road Chengdu 610041 People’s Republic of China Tel: +86 28 81801181 Fax: +86 28 85502570 E-mail: [email protected]
Research Paper Congenital Craniofacial Anomalies
Nucleofection is highly efficient for transfecting genes into murine embryonic palatal mesenchymal cells in primary culture
W.-L. Xiao1,2, B. Shi1, Q. Zheng1, Y. Wang1, L. Huang1, S. Li1, Y. Lu1, M. Wu3 1 Department of Oral and Maxillofacial Surgery, West China College of Stomatology, Sichuan University, Chengdu, People’s Republic of China; 2Department of Oral and Maxillofacial Surgery, The Medical School Hospital of Qingdao University, Qingdao, People’s Republic of China; 3Department of Biochemistry & Molecular Biology, University of North Dakota, Grand Forks, ND, USA
W.-L. Xiao, B. Shi, Q. Zheng, Y. Wang, L. Huang, S. Li, Y. Lu, M. Wu: Nucleofection is highly efficient for transfecting genes into murine embryonic palatal mesenchymal cells in primary culture. Int. J. Oral Maxillofac. Surg. 2007; 36: 429–434. # 2007 Published by Elsevier Ltd on behalf of International Association of Oral and Maxillofacial Surgeons. Abstract. Non-syndromic cleft of the lip and/or palate is one of the most common birth defects in humans. Embryonic palatal mesenchymal (EPM) cells are an attractive source for investigating embryonic palatal development. In this study, we developed a highly efficient transfection method for murine EPM (MEPM) cells. MEPM cells were transfected with the plasmid pEGFP-N1 using two non-viral methods: nucleofection and lipofection. Nucleofection provided a much better rate of gene transfer than lipofection particularly in MEPM cells. The methylenetetrahydrofolate reductase (MTHFR) gene is an important candidate for involvement in the pathogenesis of this birth defect. The RNA interference plasmid of MTHFR was constructed and nucleofected into MEPM cells. Successful transfection resulted in a remarkable reduction in the expression of MTHFR. Taken together, the results indicate that nucleofection is highly efficient for MEPM cell transfection, and that this approach may be useful for investigating gene function in the process of palatogenesis.
The etiology of non-syndromic cleft of the lip and/or palate (NCLP) is thought to be multifactorial, including genetic and environmental factors21. Embryonic palatal mesenchymal (EPM) cells are derived from embryonic palate. Primary culture of murine and human EPM cells has been described14,17,32,33 and used extensively to 0901-5027/050429 + 06 $30.00/0
investigate the mechanism of the interaction between genetic and environmental factors in the process of palatogenesis8,34. RNA interference (RNAi) as a strategy for reducing RNA expression has been proven to be a powerful tool for reverse genetic analysis20. RNAi has also shown great promise in dissecting the pathogenic
Keywords: cleft palate; RNA interference; mouse embryo; palatal mesenchymal cells; MTHFR; transfection. Accepted for publication 11 December 2006
mechanisms of many organisms11. To obtain meaningful results, this technique requires significant knockdown of a target gene. Many different approaches have been used for transfecting exogenous DNAs into primary culture cells2,3,6, but transfection with EPM cells is rarely explored
# 2007 Published by Elsevier Ltd on behalf of International Association of Oral and Maxillofacial Surgeons.
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and no satisfactory methods are currently available. Lipofectin is commonly used for transfection of RNAi, but with low efficiency. Viral vectors and electroporation may be more efficient but for technical reasons their use has often been limited. Viral vectors introduce an exogenous DNA in the form of viral genes that can complicate the analysis of gene expression, and the vectors are sometimes inactivated through methylation. Electroporation conditions that elicit the highest gene expression may also yield more cell death24. The recent development of NucleofactorTM technology allows directed electroporation of an exogenous DNA to the nucleus, and this technology has been applied successfully in transfection of primary culture cells that are generally difficult to transfect18,35. Here is described a novel nucleofection medium and conditions for reproducible transient delivery of exogenous pEGFPN1 to murine EPM (MEPM) cells. Importantly, the method was used to show knockdown of an endogenous gene, methylenetetrahydrofolate reductase (MTHFR), using a specific RNAi plasmid. The novel transfection protocol may allow delineation of gene function in MEPM cells, which was previously not feasible due to inability to achieve high efficiency transfection in this cell type. Materials and methods Cell culture
C57BL/6J mice aged 8 weeks (Jackson Laboratory, Bar Harbor, MI, USA) were maintained at a temperature of 22 8C with an alternating light/dark cycle and were given access to food and filtered water. Mature male and female (8 weeks old) were mated overnight and the presence of a vaginal plug the following morning was taken as evidence of mating (gestation day 0, GD0). Palate shelves were dissected from GD13 embryos by using a surgical microscope, as described previously3. Removed palatal shelves were washed three times with Ca–Mg free 0.01 M phosphate-buffered saline (PBS), and incubated with dispase (Gibco BRL, Life Technologies, Grand Island, NY, USA) for 18 h at 4 8C. An operation was performed according to the method described previously28 to separate the palatal mesenchyme and epithelia. The purified palatal mesenchymes was incubated with 0.25% trypsin, 0.02% EDTA in Ca–Mg free PBS for 15 min at 37 8C to dissociate individual cells. The action of trypsin was inhibited
by 4 8C Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s Medium F12 (1:1) containing 10% fetal bovine serum (FBS). The cell number was then determined using a hemocytometer. Primary cultures of MEPM cells were initiated by seeding 5 105 cells/5 ml DMEM/F12 with 10% FBS into a 25-ml tissue-culture flask. Confluent MEPM cells were subcultured at a ratio of 1:3. Transfection of pEGFP-N1 by nucleofection and lipofection
The pEGFP-N1 plasmid was purchased from Clontech (Palo Alto, CA, USA) and used to appreciate the transfection efficiency. All experiments for transfection were repeated three times. The primary culture of MEPM cells was transfected using Amaxa nucleofectorTM technology (Amaxa, Koeln, Germany). Three nucleofection programs, A23, G16, and U30, were selected to test the transfection efficiency in MEPM cells. The cells (1 106) were resuspended in solution from nucleofector Kit R, also available as part of the Amaxa cell optimization kit, following the manufacturer’s instructions for cell transfection. The cell suspension was mixed with 5 mg purified pEGFP-N1 and nucleofected using nucleofection program A23, G16 or U30. After transfection, the cells were transferred to a six-well plate with 2 ml DMEM/F12 complete media. The percentage of GFP-positive MEPM cells was assessed at 48 h after transfection using a fluorescence microscope. For comparison, pEGFP-N1 was transfected to MEPM cells by lipofection. The plasmid was mixed with Lipofectamine 2000TM according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). Briefly, for each transfection, 10 ml Lipofectamine 2000 was diluted with 250 ml serum-free OptiMEM and kept at room temperature for 5 min, mixed with 250 ml serum-free Opti-MEM containing 4 mg pEGFP-N1 plasmid DNA, and the mixture was then left at room temperature for 20 min. Prior to transfection, cells in six-well plates were rinsed twice with serum-free Opti-MEM. The above DNA–Lipofectamine 2000 complex was added to the MEPM cells. After incubation at 37 8C for 12 h, the transfection medium was replaced with fresh DMEM/F12 complete medium. The percentage of GFPpositive MEPM cells was assessed at 48 h after transfection using a fluorescence microscope.
Transfection efficiency analysis by fluorescence microscopy
The transfected MEPM cells were viewed for fluorescent protein expression using a fluorescence microscope (Olympus IX50, Olympus, Japan) equipped with phase-contrast illumination, and photographs were captured directly using a digital camera. Nine areas were randomly captured for each well in order to obtain 200 cells for analysis. The total number of MEPM cells within the areas was determined using phase contrast. Green fluorescent protein (GFP)-positive MEPM cells were also counted in the same areas. This allowed calculation of the percentage of GFP-positive MEPM cells compared to the total number of cells in all nine areas as described previously19. The percentage of GFP-positive MEPM cells represents the transfection efficiency. Construction RNAi plasmid of MTHFR gene
Four small iRNA (siRNA) sequences were designed according to the cDNA of MTHFR in GenBank (GenBank accession No. AK030192)9. The sense sequences were: S-1 GGCCT ACCTC GAATT CTTC; S-2 GAGCT ACATC TACCG CACA; S-3 CCAGC CTGAT GAAGG AAGA; S-4 GGATG TAATT GAGCC CATC. The four siRNAs suppressed mRNA expression of the MTHFR gene using the LineSilence RNATM kit (Allele Biotechnology & Pharmaceuticals, San Diego, CA, USA) as described previously7. A semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay showed that the S-3 sequence was the most efficient at screening mRNA expression of the MTHFR gene. The shRNA (short-hairpin RNA) sequence was synthesized according to S-3 and cloned into psiRNA-Hh1neo_G2 (psiRNA-MTHFR) (InvivoGen, San Diego, CA, USA). The corresponding scrambled sequence of S-3 (in which all of the bases were randomly aligned) was analysed using BLAST to eliminate the possibility of anyone genes. The shRNA sequence was then synthesized according to the nonsense strand sequence of S-3, and cloned into psiRNA-Hh1neo_G2 (psiRNA-NMTHFR). Transfection of RNAi plasmids targeting the MTHFR gene to MEPM cells
MEPM cells were nucleofected with psiRNA-Hh1neo_G2, psiRNA-MTHFR or psiRNA-NMTHFR by nucleofection
Efficiency of nucleofection in transfecting genes program U30 using the solution from the nucleofector Kit R (Amaxa). After nucleofection, the cells were seeded in six-well plates to culture for 48 h before analysing transfection efficiency.
Quantitative real-time PCR
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Statistical analysis
Results
Data analysis was performed using the SPSS statistics software package. All the results were expressed as means SEM and n refers to the number of experiments. P < 0.05 was considered statistically significant.
The MEPM cells transfected by nucleofection and lipofection with pEGFP-N1 vector were analysed by fluorescence microscopy 48 h after transfection (Fig. 1). The number of GFP-expressing cells (92%) indicated very high levels of
Total RNA was extracted by Trizol reagent (Gibco BRL) according to the manufacturer’s instructions. RNA was reversely transcribed to cDNA using a kit (Promega, Madison, WI). Real-time PCR reactions were carried out using a real-time PCR kit (Takara, Japan). The levels of GAPDH mRNA were quantified as an internal control. The RT mixture was diluted to 100 ml with glass-distilled water. The PCR products were detected using a real-time system FTC 2000 (Funglyn, Toronto, Canada). PCR reactions were performed in triplicate for each sample and the mean value at which the PCR product crossed the threshold (Ct) was calculated. The ratio of MTHFR mRNA expression to that of the internal standard mRNA (GAPDH) was calculated using a method described previously23.
Western blotting
Cells were harvested at different time points for protein extraction with mammalian protein extraction reagent (M-PER; Pierce, Rockford, IL, USA). Equal amounts of total protein were electrophoresed in a 10% SDS–PAGE gel, and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA) in a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, Richmond, CA, USA) at 15 V for 30 min. The membrane was blocked for 2 h at room temperature with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TTBS) and incubated overnight with primary polyclonal antiMTHFR antibodies (Santa Cruz, CA, USA) (1:2000 dilution), monoclonal anti-b-actin antibody (Sigma-Aldrich, St Louis, MO, USA) (1:5000 dilution). The membrane was then probed with secondary horse radish peroxidase-conjugated anti-goat antibodies (1:5000 dilution in TTBS; Santa Cruz) for 1 h and developed with an ECL Detection kit (Amersham, Uppsala, Sweden) according to the manufacturer’s instructions. The protein was detected with an ECL system (Amersham) by chemoluminescence, and visualized on radiographic film. Protein expression was quantified with Gel-Pro Analyzer 3.1 software (Media Cybernetics, Silver Spring, MD, USA).
Fig. 1. Transfection efficiency of nucleofection and lipofection for MEPM cells using pGEFPN1 analyzed by fluorescence microscopy at 24 h after transfection. A, C, E, G show phase contrast and B, D, F, H show fluorescent pictures (magnification: 100). MEPM cells were nucleofected using program A23 (A and B), G16 (C and D) or U30 (E and F). MEPM cells (G and H) were transfected using Lipofectamine 2000. The highest efficiency of transfection was obtained with nucleofection program U30. The efficiency was respectively 60 and 42% using program G16 and A23. Efficiency using lipofection was only 15%.
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Fig. 2. Significant reduction in MTHFR mRNA expression by psiRNA-MTHFR transfection. Primary cultures of MEPM cells (1 106 cells) were transfected using program U30 and the solution from nucleofector Kit R with 4 mg highly purified plasmid DNA. psiRNA-MTHFR: RNAi vector; psiRNA-NMTHFR: RNAi nonsense strands vector; psiRNA: psiRNA-Hh1neo_G2 alone vector; control: blank control. Real-Time PCR results of MTHFR mRNA expression after MTHFR gene silencing for 48 h. MTHFR mRNA expression was suppressed significantly by psiRNA-MTHFR compared to control (n = 3,*P < 0.001).
fluorescence in the nucleofected cells using nucleofection program U30. The number of GFP-expressing cells was 60% and 42% using nucleofection programs G16 and A23, respectively. All three nucleofection programs for MEPM cells did not result in any visible toxic side effects. In contrast, lipofection resulted in fewer cells with GFP expression (15%). An RNAi plasmid targeting the endogenous MTHFR gene was nucleofected to MEPM cells for studying MTHFR genesilencing efficiency. The constructed RNAi plasmid was nucleofected to MEPM cells using the most efficient nucleofection method (nucleofection program U30 and the solution from nucleofector Kit R). Using real-time PCR, it was found that the mRNA level of MTHFR was reduced by 88% at 48 h (P < 0.001) compared to control (Fig. 2). Western
blotting analysis demonstrated a significant reduction (80%) of MTHFR protein expression in MEPM cells 48 h after nucleofection compared to control (P < 0.001, Fig. 3). Discussion
EPM cells derived from embryonic palate form the bone, muscular and adipose tissue of the palate26, and these cells could be identified by staining with vimentin, S100 and desmin32. Vimentin is used as a marker of mesenchymal tissue31, S-100 protein is used as a marker of neural tissue1 and desmin is a marker of muscle tissue25. EPM cells thus have the potential to differentiate into a variety of cells with mesenchymal, neural and myoblastoid characteristics. Three nucleofection programs were selected according to the
Fig. 3. Decrease in MTHFR protein expression by western blotting analysis. Transfection was performed as above described. MTHFR protein was about 74KD and the expression levels of MTHFR protein were suppressed significantly after MTHFR gene silencing for 48 h. An anti-bactin antibody was used as an internal control. Lanes a–d: (a) control, (b) psiRNA-NMTHFR, (c) psiRNA, (d) psiRNA-MTHFR. MTHFR protein of MTHFR gene silencing for 48 h was quantitated by densitometric analysis (n = 3). *P < 0.001, indicating significant inhibition compared to control.
above characteristics of EPM cells. The highest efficiency of transfection was obtained with the program U30 without significant cell toxicity. Although lipofection has been used to transfect plasmid to EPM cells for investigating the mechanism of palatogenesis14, the transfection efficiency was unknown. The transfection efficiency of lipofection in the present study was much lower than that of nucleofection. The etiology of NCLP is thought to be multifactorial, with both genetic and environmental factors playing a role. The MTHFR gene is located on chromosome 1q36 and is a key enzyme in folic acid metabolism13. C677T (alanine to valine) was the first common MTHFR variant identified to correlate with reduced enzyme activity and increased thermolability in both the heterozygous and homozygous states12. A second common MTHFR variant, A1298C (glutamate to alanine), has also been associated with decreased enzyme activity, although to a lesser extent than C677T29. Mothers carrying the MTHFR 677TT genotype who either did not use folic acid supplements periconceptionally or had a low dietary folate intake, or both, had an increased risk of delivering a CLP child by almost sixfold, three-fold and 10-fold, respectively30. No folate supplement use, low dietary folate intake and maternal MTHFR 1298CC genotype increased the risk of CLP offspring almost seven-fold30. Some epidemiological studies also indicated that the interaction between the MTHFR gene and folic acid was a key factor during palatogenesis. The present study confirmed that the RNAi plasmid of the MTHFR gene could be nucleofected efficiently into MEPM cells, and that the expression of MTHFR in MEPM cells was remarkably inhibited. The nucleofection protocol may be used to investigate the mechanism of folic acid supplements by preventing the teratogenic effect of MTHFR mutation in cultured MEPM cells. The nucleofection protocol may also be applied to analyse the pathogenesis of the other candidate genes in NCLP, a number of which (MSX1, PVRL1, TGFb, etc.) have been identified in recent years4,10. With regard to a functional gene, conventional antisense RNA and gene knockout are the all-purpose methods of reverse genetics5,22. Gene knockout could eliminate completely the activity of a target gene while not necessarily reflecting the specific function of the gene at a particular embryonic developmental stage. The antisense RNA technology is not sufficient to
Efficiency of nucleofection in transfecting genes suppress the expression of an endogenous gene and results in a transitional phenotype, misleading our interpretation of the function of a particular gene. The ability of RNAi to efficiently suppress target genes in mammals has been heralded as the most powerful reverse genetics approach in recent years. Up to now, the mechanism of palatogenesis has been studied from the cell to organ level16,27. To further understand the role of candidate gene variants in palatogenesis, RNAi may be a useful tool. By nucleofecting the RNAi plasmid of different candidate genes into MEPM cells, the inherent mechanism of the genes resulting in cleft palate could be elucidated. It will also be worthwhile to nucleofect RNAi plasmid of different candidate genes into cultured embryonic palatal shelves of animal models to investigate the mechanisms of palatal shelf confluence at the organ level. If these candidate genes function and the cross-talk between them is elucidated, it will be possible to prenatally diagnose NCLP and reduce its incidence by preventive treatment. Transfection of primary cells is challenging15. Transfection of EPM cells has rarely been studied, and without much success. Lipofectin is commonly used for transfection of RNAi, but with low efficiency. Viral vectors and electroporation may be more efficient but for technical reasons their use has often been limited. The newer method of nucleofection is simple and has proven to be efficient in transfecting various primary cells18,35. In the present study, it was shown that the nucleofection of MEPM cells provided a method for rapid and efficient transfection. The Amaxa transfection system, with nucleofection program U30 and the solution from nucleofector Kit R, was a very efficient way to transfect the MEPM cells. This method may be useful for delineating the function of candidate genes during palatogenesis by using RNAi technology. Acknowledgement. This work was supported by grants from the National Natural Science Foundation of China (30371552).
References 1. Anneren G, Esscher T, Larsson L, Olsen L, Pahlman S. S-100 protein and neuron-specific enolase in amniotic fluid as markers of abdominal wall and neural tube defects in the foetus. Prenat Diagn 1988: 8: 323–328.
2. Behr JP, Demeneix B, Loeffler JP, Perez-Mutul J. Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc Natl Acad Sci U S A 1989: 86: 6982– 6986. 3. Bierhuizen MF, Westerman Y, Visser TP, Wognum AW, Wagemaker G. Green fluorescent protein variants as markers of retroviral-mediated gene transfer in primary hematopoietic cells and cell lines. Biochem Biophys Res Commun 1997: 234: 371–375. 4. Carinci F, Pezzetti F, Scapoli L, Martinelli M, Avantaggiato A, Carinci P, Padula E, Baciliero U, Gombos F, Laino G, Rullo R, Cenzi R, Carls F, Tognon M. Recent developments in orofacial cleft genetics. J Craniofac Surg 2003: 14: 130–143. 5. Chen Z, Karaplis AC, Ackerman SL, Pogribny IP, Melnyk S, LussierCacan S, Chen MF, Pai A, John SW, Smith RS, Bottiglieri T, Bagley P, Selhub J, Rudnicki MA, James SJ, Rozen R. Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition. Hum Mol Genet 2001: 10: 433–443. 6. Chu G, Hayakawa H, Berg P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res 1987: 15: 1311–1326. 7. Csiszar A, Ungvari Z, Koller AG, Edwards J, Kaley G. Proinflammatory phenotype of coronary arteries promotes endothelial apoptosis in aging. Physiol Genomics 2004: 17: 21–30. 8. Dhulipala VC, Hanumegowda UM, Balasubramanian G, Reddy CS. Relevance of the palatal protein kinase A pathway to the pathogenesis of cleft palate by secalonic acid D in mice. Toxicol Appl Pharmacol 2004: 194: 270–279. 9. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21and 22-nucleotide RNAs. Genes Dev 2001: 15: 188–200. 10. Farrall M, Holder S. Familial recurrence-pattern analysis of cleft lip with or without cleft palate. Am J Hum Genet 1992: 50: 270–277. 11. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998: 391: 806–811. 12. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995: 10: 111–113. 13. Goyette P, Pai A, Milos R, Frosst P, Tran P, Chen Z, Chan M, Rozen R. Gene structure of human and mouse
14.
15.
16.
17.
18.
19.
20.
21. 22.
23.
24.
25.
26.
433
methylenetetrahydrofolate reductase (MTHFR). Mamm Genome 1998: 9: 652–656. Greene RM, Nugent P, Mukhopadhyay P, Warner DR, Pisano MM. Intracellular dynamics of Smad-mediated TGFbeta signaling. J Cell Physiol 2003: 197: 261–271. Gresch O, Engel FB, Nesic D, Tran TT, England HM, Hickman ES, Korner I, Gan L, Chen S, Castro-Obregon S, Hammermann R, Wolf J, Muller-Hartmann H, Nix M, Siebenkotten G, Kraus G, Lun K. New nonviral method for gene transfer into primary cells. Methods 2004: 33: 151–163. Han J, Xiao Y, Lin J, Li Y. PDGF-C Controls proliferation and is down-regulated by retinoic acid in mouse embryonic palatal mesenchymal cells. Birth Def Res B Dev Reprod Toxicol 2006: 77: 438– 444. Hanumegowda UM, Dhulipala VC, Reddy CS. Mechanism of secalonic acid D-induced inhibition of transcription factor binding to cyclic AMP response element in the developing murine palate. Toxicol Sci 2002: 70: 55–62. Lakshmipathy U, Pelacho B, Sudo K, Linehan JL, Coucouvanis E, Kaufman DS, Verfaillie CM. Efficient transfection of embryonic and adult stem cells. Stem Cells 2004: 22: 531–543. Leclere PG, Panjwani A, Docherty R, Berry M, Pizzey J, Tonge DA. Effective gene delivery to adult neurons by a modified form of electroporation. J Neurosci Methods 2005: 142: 137– 143. Morris JC, Wang Z, Drew ME, Englund PT. Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library. EMBO J 2002: 21: 4429–4438. Murray JC. Gene/environment causes of cleft lip and/or palate. Clin Genet 2002: 61: 248–256. O’Brodorich H. Fetal lung liquid secretion: insights using the tools of inhibitors and genetic knock-out experiments. Am J Respir Cell Mol Biol 2001: 25: 8–10. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002: 30: e36. Pinero J, Lopez-Baena M, Ortiz T, Cortes F. Apoptotic and necrotic cell death are both induced by electroporation in HL60 human promyeloid leukaemia cells. Apoptosis 1997: 2: 330–336. Schaart G, Viebahn C, Langmann W, Ramaekers F. Desmin and titin expression in early postimplantation mouse embryos. Development 1989: 107: 585– 596. Sperber GH, Machin GA. The enigma of cephalogenesis. Cleft Palate Craniofac J 1994: 31: 91–96.
434
Xiao et al.
27. Sun D, Vanderburg CR, Odierna GS, Hay ED. TGFbeta3 promotes transformation of chicken palate medial edge epithelium to mesenchyme in vitro. Development 1998: 125: 95–105. 28. Tyler MS, Pratt RM. Effect of epidermal growth factor on secondary palatal epithelium in vitro: tissue isolation and recombination studies. J Embryol Exp Morphol 1980: 58: 93–106. 29. van der Put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes TK, van den Heuvel LP, Blom HJ. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet 1998: 62: 1044–1051. 30. van Rooij IA, Vermeij-Keers C, Kluijtmans LA, Ocke MC, Zielhuis GA, Goorhuis-Brouwer SM, van der Biezen JJ, Kuijpers-Jagtman AM, Steegers-Theunissen RP. Does the interaction between maternal folate
31.
32.
33.
34.
intake and the methylenetetrahydrofolate reducetase polymorphisms affect the risk of cleft lip with or without cleft palate? Am J Epidemiol 2003: 157: 583–591. von Koskull H, Virtanen I. Induction of cytokeratin expression in human mesenchymal cells. J Cell Physiol 1987: 133: 321–329. Yano H, Yoshimoto H, Ohtsuru A, Ito M, Yamashita S, Fujii T. Characterization of cultured rat embryonic palatal mesenchymal cells. Cleft Palate Craniofac J 1996: 33: 379–384. Yoneda T, Urade M, Sakuda M, Miyazaki T. Altered growth, differentiation, and responsiveness to epidermal growth factor of human embryonic mesenchymal cells of palate by persistent rubella virus infection. J Clin Invest 1986: 77: 1613– 1621. Yu Z, Xing Y. All-trans retinoic acid inhibited chondrogenesis of mouse embryonic palate mesenchymal cells by down-regulation of TGF-beta/Smad sig-
naling. Biochem Biophys Res Commun 2006: 340: 929–934. 35. Zernecke A, Erl W, Fraemohs L, Lietz M, Weber C. Suppression of endothelial adhesion molecule up-regulation with cyclopentenone prostaglandins is dissociated from IkappaB-alpha kinase inhibition and cell death induction. FASEB J 2003: 17: 1099–1101. Address: Bing Shi Department of Oral and Maxillofacial Surgery West China College of Stomatology of Sichuan University No. 14 Section 3 Ren Min Nan Road Chengdu 610041 People’s Republic of China Tel: +86 28 81801181 Fax: +86 28 85502570 E-mail: [email protected]