Assessing the Functional Characteristics of Synonymous and Nonsynonymous Mutation Candidates by Use of Large DNA Constructs
2007; Elsevier BV; Volume: 80; Issue: 4 Linguagem: Inglês
10.1086/513287
ISSN1537-6605
AutoresAngela Eeds, Douglas P. Mortlock, Richard Wade‐Martins, Marshall Summar,
Tópico(s)RNA Research and Splicing
ResumoAs we identify more and more genetic changes, either through mutation studies or population screens, we need powerful tools to study their potential molecular effects. With these tools, we can begin to understand the contributions of genetic variations to the wide range of human phenotypes. We used our catalogue of molecular changes in patients with carbamyl phosphate synthetase I (CPSI) deficiency to develop such a system for use in eukaryotic cells. We developed the tools and methods for rapidly modifying bacterial artificial chromosomes (BACs) for eukaryotic episomal replication, marker expression, and selection and then applied this protocol to a BAC containing the entire CPSI gene. Although this CPSI BAC construct was suitable for studying nonsynonymous mutations, potential splicing defects, and promoter variations, our focus was on studying potential splicing and RNA-processing defects to validate this system. In this article, we describe the construction of this system and subsequently examine the mechanism of four putative splicing mutations in patients deficient in CPSI. Using this model, we also demonstrate the reversible role of nonsense-mediated decay in all four mutations, using small interfering RNA knockdown of hUPF2. Furthermore, we were able to locate cryptic splicing sites for the two intronic mutations. This BAC-based system permits expression studies in the absence of patient RNA or tissues with relevant gene expression and provides experimental flexibility not available in genomic DNA or plasmid constructs. Our splicing and RNA degradation data demonstrate the advantages of using whole-gene constructs to study the effects of sequence variation on gene expression and function. As we identify more and more genetic changes, either through mutation studies or population screens, we need powerful tools to study their potential molecular effects. With these tools, we can begin to understand the contributions of genetic variations to the wide range of human phenotypes. We used our catalogue of molecular changes in patients with carbamyl phosphate synthetase I (CPSI) deficiency to develop such a system for use in eukaryotic cells. We developed the tools and methods for rapidly modifying bacterial artificial chromosomes (BACs) for eukaryotic episomal replication, marker expression, and selection and then applied this protocol to a BAC containing the entire CPSI gene. Although this CPSI BAC construct was suitable for studying nonsynonymous mutations, potential splicing defects, and promoter variations, our focus was on studying potential splicing and RNA-processing defects to validate this system. In this article, we describe the construction of this system and subsequently examine the mechanism of four putative splicing mutations in patients deficient in CPSI. Using this model, we also demonstrate the reversible role of nonsense-mediated decay in all four mutations, using small interfering RNA knockdown of hUPF2. Furthermore, we were able to locate cryptic splicing sites for the two intronic mutations. This BAC-based system permits expression studies in the absence of patient RNA or tissues with relevant gene expression and provides experimental flexibility not available in genomic DNA or plasmid constructs. Our splicing and RNA degradation data demonstrate the advantages of using whole-gene constructs to study the effects of sequence variation on gene expression and function. Identifying genetic contributions to a disease phenotype requires an understanding of the molecular pathology of mutations. Genetic variants can alter protein function through amino acid substitutions and promoter function and sensitivity through alterations in binding sites. Additionally, changing intronic and exonic sequences may result in splicing or RNA-processing defects.1Faustino NA Cooper TA Pre-mRNA splicing and human disease.Genes Dev. 2003; 17: 419-437Crossref PubMed Scopus (985) Google Scholar, 2Nissim-Rafinia M Kerem B The splicing machinery is a genetic modifier of disease severity.Trends Genet. 2005; 21: 480-483Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Such RNA-processing defects often trigger the nonsense-mediated decay (NMD) pathway, a surveillance mechanism that degrades mRNA transcripts encoding a premature termination codon (PTC).3Maquat LE Nonsense-mediated mRNA decay in mammals.J Cell Sci. 2005; 118: 1773-1776Crossref PubMed Scopus (223) Google Scholar It is estimated that NMD accounts for as many as 30% of disease alleles.4Frischmeyer PA Dietz HC Nonsense-mediated mRNA decay in health and disease.Hum Mol Genet. 1999; 8: 1893-1900Crossref PubMed Scopus (850) Google Scholar, 5Mendell JT Dietz HC When the message goes awry: disease-producing mutations that influence mRNA content and performance.Cell. 2001; 107: 411-414Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar When molecular variation in a gene is under investigation, it is imperative but often difficult to determine possible functionality and biological relevance. Specifically, RNA-processing defects are often difficult to study without the development of elaborate artificial constructs. We recently examined the impact of RNA-processing defects that trigger NMD in patients with carbamyl phosphate synthetase I deficiency (CPSID [MIM #237300]).6Eeds A Hall LD Yadav M Willis AS Summar S Putnam A Barr F Summar ML The frequent observation of evidence for nonsense-mediated decay in RNA from patients with carbamyl phosphate synthetase I deficiency.Mol Genet Metab. 2006; 89: 80-86Crossref PubMed Scopus (15) Google Scholar CPSID is an autosomal recessive disease characterized by hyperammonemia and is the result of defects in carbamyl phosphate synthetase I (CPSI), the enzyme required to catalyze the first and rate-determining step of the hepatic urea cycle. We found RNA-based physical evidence that ∼40% of the novel mutations resulting in CPSID created RNA-processing defects that trigger NMD.6Eeds A Hall LD Yadav M Willis AS Summar S Putnam A Barr F Summar ML The frequent observation of evidence for nonsense-mediated decay in RNA from patients with carbamyl phosphate synthetase I deficiency.Mol Genet Metab. 2006; 89: 80-86Crossref PubMed Scopus (15) Google Scholar Unfortunately, the unavailability of cells that expressed the CPSI gene in both adequate quantity and quality hampered our study. Consequently, we were forced to eliminate almost 2/3 of the patients for whom only genomic DNA (gDNA) was available. Examining the functional mechanisms of pathogenic alleles in many diseases such as CPSID presents a formidable challenge due to the frequent lack of patient tissue for RNA analysis, as well as to the large size of the gene, which has 38 exons that span >120 kb of gDNA.7Summar ML Hall LD Eeds AM Hutcheson HB Kuo AN Willis AS Rubio V Arvin MK Schofield JP Dawson EP Characterization of genomic structure and polymorphisms in the human carbamyl phosphate synthetase I gene.Gene. 2003; 311: 51-57Crossref PubMed Scopus (46) Google Scholar Developing the means to explore these functional mechanisms is absolutely crucial for determining both the potential pathologic effects of rare variations and the milder functional variation seen in common polymorphisms. This problem is not unique to the study of CPSI mutations; indeed, it will certainly warrant considerable attention as more genetic changes become associated with a variety of common disease states. To facilitate functional genetics studies, we developed a versatile BAC-based model system to test the effects of a diverse set of intronic and exonic mutations in a CPSI whole-gene construct. Essential elements of this system included normal BAC replication in bacteria, episomal eukaryotic replication, antibiotic selection in eukaryotic cells, and the expression of green fluorescent protein (GFP). Each element was incorporated into a vector that is easily exported to other BAC constructs. This BAC-based expression system, coupled with other recent BAC engineering and mutagenesis improvements,8Hart SL Rancibia-Carcamo CV Wolfert MA Mailhos C O'Reilly NJ Ali RR Coutelle C George AJ Harbottle RP Knight AM et al.Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector.Hum Gene Ther. 1998; 9: 575-585Crossref PubMed Scopus (180) Google Scholar, 9Warming S Costantino N Court DL Jenkins NA Copeland NG Simple and highly efficient BAC recombineering using galK selection.Nucleic Acids Res. 2005; 33: e36Crossref PubMed Scopus (937) Google Scholar provides an efficient way to test genetic variants, irrespective of type, location, or gene size. This expression system is widely applicable to many experiments that examine both coding and noncoding sequences, and it is particularly useful for assaying mutations that affect RNA processing because of the large size of genomic inserts allowed by BACs. Additionally, studying mutations within their wider sequence context should enable us to determine their mechanistic effect with greater accuracy. Here, we describe the construction and testing of this system and demonstrate its utility as an effective eukaryotic cell model in the study of the role of genetic variations with both known and unknown molecular effects identified in patients with CPSID. We inserted the pEHG plasmid into the backbone of BAC RP11-349G4, using Cre/loxP recombination, as described elsewhere.10Wade-Martins R Smith ER Tyminski E Chiocca EA Saeki Y An infectious transfer and expression system for genomic DNA loci in human and mouse cells.Nat Biotechnol. 2001; 19: 1067-1070Crossref PubMed Scopus (158) Google Scholar To provide ubiquitous expression of the normally hepatically expressed CPSI in our test cells (MRC-5V2 cells),11Huschtscha LI Holliday R Limited and unlimited growth of SV40-transformed cells from human diploid MRC-5 fibroblasts.J Cell Sci. 1983; 63: 77-99Crossref PubMed Google Scholar we inserted the human cytomegalovirus (CMV) immediate-early promoter (Towne strain) upstream of CPSI. We modified pCMV/Bsd (Invitrogen) by inserting a KpnI site at position 1937 by site-directed mutagenesis (Stratagene QuickChange). Then, KpnI and XhoI were used to create a complementary insertion site for an oligo containing the tetracycline resistance cassette flanked by FRT sites from plasmid pTet/FRT.12Mortlock DP Guenther C Kingsley DM A general approach for identifying distant regulatory elements applied to the Gdf6 gene.Genome Res. 2003; 13: 2069-2081Crossref PubMed Scopus (82) Google Scholar A 5′ homology arm (Invitrogen) containing 50 bp of sequence homologous to the BAC backbone was then synthesized with NotI and NheI overhangs to direct insertion of the homology arm immediately 5′ of the Tet/FRT sequence. A 3′ homology arm (Invitrogen) containing 50 bp of sequence homologous to the CPSI gene was synthesized, beginning with the transcription start site of CPSI (AF154830), with RsrII and XmaI overhangs to direct insertion of the homology arm downstream of the CMV promoter. The BAC+pEHG construct was transformed into EL250 Escherichia coli cells, and a monoclonal colony was induced for recombination by a temperature shift from 32°C to 42°C before electroporation of the constructed CMV vector.13Lee EC Yu D Martinez de Velasco J Tessarollo L Swing DA Court DL Jenkins NA Copeland NG A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.Genomics. 2001; 73: 56-65Crossref PubMed Scopus (997) Google Scholar Recombinants were selected with tetracycline, after which the tetracycline cassette was removed by arabinose induction of Flp recombinase. Site-directed mutagenesis to introduce point mutations into the wild-type (wt) BECC (BAC+pEHG+CMV+CPSI) construct was performed using double homologous recombination with galK selection after transfection of the wtBECC construct into SW102 cells.9Warming S Costantino N Court DL Jenkins NA Copeland NG Simple and highly efficient BAC recombineering using galK selection.Nucleic Acids Res. 2005; 33: e36Crossref PubMed Scopus (937) Google Scholar The first homologous recombination step is to add the galK gene at the position of the desired mutation, with the use of positive selection for colonies able to metabolize galactose. The second homologous recombination step is to replace the galK cassette with the desired mutation sequence, with the use of negative selection against colonies that retain galK and therefore toxically metabolize 2-deoxygalactose, which produces a toxin in the presence of galK expression. All restriction enzymes were purchased from New England Biolabs. BAC purifications were performed using the NucleoBond BAC Maxi AX500 kit (BDBiosciences). Pulse-field gel electrophoresis (PFGE) and fingerprinting revealed no unwanted or global rearrangements of the vector during the retrofitting or homologous recombination steps. PFGE was performed after a 3-h digestion of BAC DNA with NotI (New England Biolabs) in a 1% agarose 0.5% tris-acetate EDTA gel run at 6 V for 16 h, with an initial switching time of 0.2 s ramped to a final switching time of 22 s. Fingerprinting was performed by digesting BAC DNA with BamHI (New England Biolabs) for 3 h and then running the samples on a 1% agarose 1% tris-borate EDTA gel for 16 h at 35 V. Direct sequencing was also performed to verify proper insertion of the CMV promoter upstream of CPSI. Single-strand conformation polymorphism (SSCP) analysis for mutation detection in the coding region of CSPI revealed that no unwanted rearrangements or mutations occurred in the coding region of the gene during construct manipulations with the use of a previously published protocol and primer pairs.7Summar ML Hall LD Eeds AM Hutcheson HB Kuo AN Willis AS Rubio V Arvin MK Schofield JP Dawson EP Characterization of genomic structure and polymorphisms in the human carbamyl phosphate synthetase I gene.Gene. 2003; 311: 51-57Crossref PubMed Scopus (46) Google Scholar, 14Willis AS Freeman ML Summar SR Barr FE Williams SM Dawson E Summar ML Ethnic diversity in a critical gene responsible for glutathione synthesis.Free Radic Biol Med. 2003; 34: 72-76Crossref PubMed Scopus (18) Google Scholar All exons, as well as the 5′ and 3′ UTRs, were screened in a total of 42 PCRs. To increase mutation detection rates, direct sequencing was performed instead of SSCP for 12 of the 42 PCRs. In addition, changes between the BECC construct and a control DNA sample detected by SSCP were analyzed by direct sequencing, and all changes were shown to be different alleles of polymorphisms reported elsewhere.7Summar ML Hall LD Eeds AM Hutcheson HB Kuo AN Willis AS Rubio V Arvin MK Schofield JP Dawson EP Characterization of genomic structure and polymorphisms in the human carbamyl phosphate synthetase I gene.Gene. 2003; 311: 51-57Crossref PubMed Scopus (46) Google Scholar Fluorescent sequencing was performed by GenHunter Corporation and the Vanderbilt University Medical Center Core Facility, with use of BigDye chemistry from Applied Biosystems. We performed radioactive dideoxynucleotide sequencing, using the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (USB) in accordance with the manufacturer's protocol, with 200 ng DNA. Transfections were performed using the LID (Lipofectin:Integrin:DNA) technique8Hart SL Rancibia-Carcamo CV Wolfert MA Mailhos C O'Reilly NJ Ali RR Coutelle C George AJ Harbottle RP Knight AM et al.Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector.Hum Gene Ther. 1998; 9: 575-585Crossref PubMed Scopus (180) Google Scholar with peptide 6. We used 8 μl lipofectin (Invitrogen), 8 μg peptide 6, and 4 μg of each BAC construct in a 2L:2I:1D ratio. Lipofectin was diluted in 400 μl Optimem (Gibco) and was incubated 45 min before the addition of peptide 6 and DNA, which was also diluted in 400 μl Optimem. Reactions were incubated for 10 min, during which time all cells were washed with 3 ml Optimem (no serum). All transfection reactions were increased to a 4-ml total volume with Optimem before dropwise placement on 5-cm dishes containing cells at 60% confluence. After a 16-h incubation, the transfection media were replaced with Dulbecco's modified Eagle medium (DMEM) and 10% fetal bovine serum for 72 h. After 72 h, hygromycin selection was added at a concentration of 125 μg/ml. After 2 wk under hygromycin selection, cells were flow-sorted for GFP expression. All GFP-positive cells were cultured under hygromycin selection as polyclonal cell lines. Cells were lysed by sonication and were quantified using the BCA Protein Estimation Kit (Pierce). After a 5-min denaturation step, equal microgram quantities of cell lysates were electrophoresed in a 6% polyacrylamide gel for 45 min at 200 V. Proteins were transferred to a nitrocellulose membrane. The anti-CPSI (product number sc-10516 [Santa Cruz]) primary antibody was incubated overnight at 4°C at a final dilution of 1:50. The bovine antigoat immunoglobulin G (IgG)–alkaline phosphatase (AP) (product number sc-2351 [Santa Cruz]) secondary antibody was incubated (1:2,000 dilution) at room temperature before alkaline phosphate staining (Bio-Rad). We used the Wizard Genomic DNA Purification Kit (Promega) to isolate DNA from all cell lines. To determine the presence of BAC DNA in transfected cell lines, we performed PCR with an upper primer complementary to the CMV promoter—UCMV (5′-CCATCCACGCTGTTTTGACCTC-3′)—and with a lower primer complementary to the sequence in the first exon of CPSI—L173 (5′-CCAGTCTTCAGTGTCCTCA-3′). The amplification thermoprofile was an initial denaturation step of 4 min at 95°C, 40 cycles of 30 s at 95°C, 30 s at 67°C, and 30 s at 72°C, and a final hold at 72°C for 10 min. To isolate RNA, we used the RNeasy Midi Kit, including the optional RNase-free DNase set, following the manufacturer's protocol (Qiagen). Reverse transcriptions were performed using 2 μg or 5 μg of total RNA and the TaqMan Reverse Transcription Reagents Kit (Applied Biosystems). The amplification thermoprofile was an initial denaturation at 65°C for 5 min, then a primer-annealing phase at 27°C for 10 min, an extension phase at 42°C for 45 min, and an enzyme denaturation step at 95°C for 5 min. We performed PCR, using the cDNA templates produced from 2 μg RNA to amplify desired regions of CPSI, for the determination of splicing changes from the intronic mutations. After visualization on a 2% agarose gel, bands were purified using the Wizard PCR Cleanup Kit (Invitrogen) and then were sequenced. PCR primers were designed at least one exon away from the mutation in question, to eliminate gDNA contamination. We amplified cDNA, using UBacT344A (5′-CTGCTCAGAATCATGACC-3′) and L1361 (5′-GGCTTCGGTAAGACTGATGT-3′) for c.1210-1G→T and U393 (5′-AGGACAGATTCTCACAATGG-3′) and L829 (5′-CTAGCAGGCGGATTACATTG-3′) for c.652-3T→G. For northern blotting, 10 μg of total RNA was used in a 1.2% agarose formaldehyde gel that was run for 2.5 h at 3 V/cm. RNA was transferred to a Hybond N+ membrane by use of the Turboblotter apparatus (Schleicher and Schuell) and was UV crosslinked. The membrane was prehybridized in Church buffer (0.25 M Na2HPO4, 7% SDS, and 50 μg/ml sheared salmon sperm DNA) at 65°C for 1 h, and hybridization occurred overnight at 65°C with either a CPSI or cyclophilin probe that was previously radiolabeled with the Prime-It RmT Random Primer Labeling Kit (Stratagene) and diluted to 1 million counts per min. We calculated relative RNA expression levels, using quantitative PCR with cDNA templates synthesized from 5 μg RNA. We performed these reactions using TaqMan (Applied Biosystems) technology on an automated platform with the ABI PRISM Detection System. To measure CPSI expression, we used the Hs00919484_m1 assay that spans the exon 3–exon 4 junction and the Hs00919480_m1 assay that spans the exon 34–exon 35 junction. We used an E-GFP probe as the endogenous control (part number 4331348 [Custom Taqman Gene Expression Assay Service]). All experiments were performed in triplicate. "No RT" samples were used as a PCR control measure for gDNA contamination. We performed ΔΔCt analysis, a description of relative RNA expression levels, by first calculating the difference between the average cycle time (Ct) value of each target CPSI sample and the average Ct value of the corresponding endogenous GFP sample at the 0.200 fluorescence threshold. Standardizing the CPSI Ct values to the GFP transcripts serves as a control, since GFP expression remains constant, and can also account for variation in BAC copy number, since both genes are present in a 1:1 ratio and are driven by a CMV promoter. These calculations were then expressed in relation to the calibrator (wild-type cell line in fig. 5A and untreated cell lines in fig. 6), which was arbitrarily set at an expression level of 100% (fig. 5A), or onefold (fig. 6).Figure 6ΔΔCt analysis of qRT-PCR data after siRNA-mediated knockdown of UPF2. Each panel graphs the relative expression levels of CPSI transcripts after UPF2 siRNA or vehicle-only treatments in the respective cell line as compared with untreated cells (set at a value of 1). Gray bars indicate relative levels measured from the exon 3–exon 4 TaqMan probe, and black bars indicate relative levels measured from the exon 34–exon 35 TaqMan probe. Error bars represent SE.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We used small interfering RNA (siRNA) to knock down UPF2 and to therefore block NMD, as performed elsewhere.15Mendell JT ap Rhys CM Dietz HC Separable roles for rent1/hUpf1 in altered splicing and decay of nonsense transcripts.Science. 2002; 298: 419-422Crossref PubMed Scopus (224) Google Scholar Cells were 75% confluent in a 10-cm dish and were treated for 3 consecutive d. SiQuest transfection reagent was used according to the manufacturer's protocol, at a final concentration of 5 μl/ml of DMEM (Mirus). siRNAs were used at a final concentration of 70 nM (Dharmacon). The siRNA sequence for UPF2 was published elsewhere,15Mendell JT ap Rhys CM Dietz HC Separable roles for rent1/hUpf1 in altered splicing and decay of nonsense transcripts.Science. 2002; 298: 419-422Crossref PubMed Scopus (224) Google Scholar and the commercially available siCONTROL RISC-Free kit (Dharmacon) used to show knockdown was specific for UPF2, since this siRNA sequence does not target any known human genes. Western blotting was performed using anti-hUPF2, anti-α-tubulin (Abcam product number ab15246), and anti-CPSI (product number sc-10516 [Santa Cruz]) primary antibodies, as well as the electrochemiluminescent rabbit IgG (Amersham NA934) and bovine anti-goat IgG-AP (product number sc-2351 [Santa Cruz]) secondary antibodies. Western blot densitometry quantification was performed on blots repeated in triplicate, with use of the QuantityOne software (BioRad). Here, we have identified the mutation mechanisms of two exonic and two intronic mutations originally identified by genomic mutation screens of patients with CPSID.6Eeds A Hall LD Yadav M Willis AS Summar S Putnam A Barr F Summar ML The frequent observation of evidence for nonsense-mediated decay in RNA from patients with carbamyl phosphate synthetase I deficiency.Mol Genet Metab. 2006; 89: 80-86Crossref PubMed Scopus (15) Google Scholar, 16Summar ML Molecular genetic research into carbamoyl-phosphate synthase I: molecular defects and linkage markers.J Inherit Metab Dis. 1998; 21: 30-39Crossref PubMed Scopus (28) Google Scholar The c.1893T→G substitution is a nonsense mutation located in exon 16, and the c.2388C→A mutation is a synonymous change in exon 19. Initially, we did not believe that this silent mutation was pathogenic. However, we were unable to identify another mutation on this patient allele. The two intronic substitutions, c.652-3T→G in intron 5 and c.1210-1G→T in intron 10, are both located at the 3′ splice acceptor site. Each mutation was unique to the patient with disease, since no others were detected in a screen of >200 additional chromosomes. To study these and other CPSI mutations in a full-gene context, we modified a BAC construct to create a platform for mutation testing. Figure 1A shows the construction of this BECC vector. First, we used Cre/loxP recombination to retrofit BAC RP11-349G4 (containing the full CPSI gene) with vector pEHG. This vector contains episomal retention elements from Epstein-Barr virus (EBV)—specifically, the latent origin of replication (OriP) and the EBV nuclear antigen 1 (EBNA-1)—as well as the hygromycin resistance antibiotic marker and a constitutive GFP reporter (E-GFP) driven by the CMV promoter.17Wade-Martins R Frampton J James MR Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells.Nucleic Acids Res. 1999; 27: 1674-1682Crossref PubMed Scopus (65) Google Scholar, 18Wade-Martins R White RE Kimura H Cook PR James MR Stable correction of a genetic deficiency in human cells by an episome carrying a 115 kb genomic transgene.Nat Biotechnol. 2000; 18: 1311-1314Crossref PubMed Scopus (69) Google ScholarFigure 1B shows a PFGE illustrating the insertion of one copy of this 11-kb vector. Second, homologous recombination allowed simultaneous deletion of the sequence 5′ of CPSI and insertion of the CMV promoter directly upstream of the first exon, thereby producing constitutive expression of this hepatic-specific gene. The CMV promoter was inserted using a Tet-CMV targeting construct flanked by BAC homology arms (fig. 1A). Multiple assays verified the integrity of this construct (see the "Materials and Methods" section). The completed wild-type construct (wtBECC) is 170 kb in size, of which 150 kb is gDNA from chromosome 2 containing the complete CPSI gene and 11 kb is from pEHG (fig. 1C). We next performed site-directed mutagenesis to introduce the c.652-3T→G, c.1210-1G→T, c.1893T→G, and c.2388C→A mutations into the BECC vector. These mutations were introduced using two-step homologous recombination with positive and negative galK selection.9Warming S Costantino N Court DL Jenkins NA Copeland NG Simple and highly efficient BAC recombineering using galK selection.Nucleic Acids Res. 2005; 33: e36Crossref PubMed Scopus (937) Google Scholar Various control assays, including PFGE and sequencing, verified that only the desired point mutations were incorporated (data not shown). We then created polyclonal, stable cell lines for each of the BECC constructs and examined expression of the exogenous genes in cultured cells. After transfection into the MRC-5V2 immortalized human lung fibroblast cell line,8Hart SL Rancibia-Carcamo CV Wolfert MA Mailhos C O'Reilly NJ Ali RR Coutelle C George AJ Harbottle RP Knight AM et al.Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector.Hum Gene Ther. 1998; 9: 575-585Crossref PubMed Scopus (180) Google Scholar, 11Huschtscha LI Holliday R Limited and unlimited growth of SV40-transformed cells from human diploid MRC-5 fibroblasts.J Cell Sci. 1983; 63: 77-99Crossref PubMed Google Scholar we obtained initial 20% transfection efficiencies as determined by visual inspection of GFP-expressing cells. GFP-positive cells demonstrating stable transfection were selected by collection through flow-sorting followed by continuous culture under hygromycin selection. The MRC+wtCPSI polyclonal cell line was passaged >85 times during a 1.5-year period under continuous hygromycin selection with no visual loss of GFP expression. To illustrate the presence of BAC-derived CPSI DNA in each cell line, we specifically amplified exogenous CPSI DNA, using the BAC-specific CMV promoter sequence and CPSI exon 1 sequence (fig. 2A). PCR revealed the presence of BAC-derived CPSI DNA only in the transfected cell lines (fig. 2B). Furthermore, we performed western blotting to verify the presence of exogenous CPSI protein specifically in the MRC+wtBECC cell line. Our results showed that, after transfection, not only did the BECC constructs create stable transformants that expressed GFP and were resistant to hygromycin, but the wild-type construct also produced CPSI (fig. 2C). To determine the effect of both intronic mutations on splicing, we performed RT-PCR on RNA isolated from the c.652-3T→G, c.1210-1G→T, and wild-type transfected cell lines. Because the reverse transcription protocol that we used was previously proved sensitive enough to pick up low-level CPSI transcripts from a nonhepatic cell line,16Summar ML Molecular genetic research into carbamoyl-phosphate synthase I: molecular defects and linkage markers.J Inherit Metab Dis. 1998; 21: 30-39Crossref PubMed Scopus (28) Google Scholar it was important to distinguish between BAC-derived and low-level endogenous MRC-5V2 CPSI transcripts. We first illustrate that the BECC model system mimics the original patient sequence data for c.652-3T→G. Whereas patient gDNA showed a heterozygous mutation at the −3 position of intron 5 (fig. 3A), patient cDNA (from a fibroblast cell line) showed a heterozygous 2-bp frameshift beginning in exon 6, indicating that the mutation activated an aberrant splice site (fig. 3B and 3E). To verify that the BECC platform reproduced what we observed directly from the patient, we performed RT-PCR on the MRC+wtCPSI and MRC+c.652-3T→G cell lines. Because a size difference indicating a splicing change from the c.652-3T→G mutation was not detectable on an agarose gel (fig. 3C), the RT-PCR products were radioactively sequenced to visualize the presence of aberrant transcript and to separate endogenous and exogenous transcripts. The sequence from the MRC+c.652-3T→G cell line mimicked the patient data by revealing the same 2-bp AG dinucleotide splicing alteration (fig. 3D and 3E). This comparison o
Referência(s)