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Genetic rearrangements of variable di-residue (RVD)-containing repeat arrays in a baculoviral TALEN system.

Lau CH, Zhu H, Tay JC, Li Z, Tay FC, Chen C, Tan WK, Du S, Sia VK, Phang RZ, Tang SY, Yang C, Chi Z, Liang CC, Ning E, Wang S - Mol Ther Methods Clin Dev (2014)

Bottom Line: The DNA sequence changes in the domains included deletion, addition, substitution, and DNA strand exchange between the left and right TALEN arms.Based on these observations, we have developed a protocol using a low MOI to produce baculoviral vectors expressing TALEN left and right arms separately.Cotransduction of the viruses produced by this optimal protocol provided an improved TALEN cleavage efficiency and enabled effective site-specific transgene integration in human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.

ABSTRACT
Virus-derived gene transfer vectors have been successfully employed to express the transcription activator-like effector nucleases (TALENs) in mammalian cells. Since the DNA-binding domains of TALENs consist of the variable di-residue (RVD)-containing tandem repeat modules and virus genome with repeated sequences is susceptible to genetic recombination, we investigated several factors that might affect TALEN cleavage efficiency of baculoviral vectors. Using a TALEN system designed to target the AAVS1 locus, we observed increased sequence instability of the TALE repeat arrays when a higher multiplicity of infection (MOI) of recombinant viruses was used to produce the baculoviral vectors. We also detected more deleterious mutations in the TALE DNA-binding domains when both left and right TALEN arms were placed into a single expression cassette as compared to the viruses containing one arm only. The DNA sequence changes in the domains included deletion, addition, substitution, and DNA strand exchange between the left and right TALEN arms. Based on these observations, we have developed a protocol using a low MOI to produce baculoviral vectors expressing TALEN left and right arms separately. Cotransduction of the viruses produced by this optimal protocol provided an improved TALEN cleavage efficiency and enabled effective site-specific transgene integration in human cells.

No MeSH data available.


Related in: MedlinePlus

Development of a TA cloning- and RE digestion-based method to examine genetic rearrangements of TALE repeat arrays. (a) Work flow chart. A fusion expression cassette bearing both left and right TALEN arms (TALEN L-R) is amplified with forward and reverse PCR primers (FP and RP) designed to amplify the full-length of tandem repeat arrays. The PCR products are inserted into a TA cloning vector. EcoRI digestion of individual clones followed by agarose gel electrophoresis is used to detect the size change of the TALE repeat arrays. The detection of a 1.76 kb DNA fragment indicates that the number of the tandem repeat units remains correct in the TALEN expression cassette. To further classify the genetic rearrangement events, the clones with no obvious size changes are subjected to DNA sequencing. (b) The use of an enzyme mixture allows PCR amplification of single band. Left: Platinum Taq DNA Polymerase High Fidelity. Right: Platinum Taq DNA Polymerase High Fidelity mixed with Elongase (1:2). (c) TA cloning and EcoRI digestion detect no change in the size of the TALE repeat arrays in plasmid vector samples. The band at 3 kb after EcoRI digestion is the linearized cloning vector, while the DNA band at 1.76 kb is the DNA insert bearing a TALE repeat array. “–“ and “+”: Without and with EcoRI. One kb DNA ladder was used as a molecular weight standard. (d) DNA sequencing analysis confirms no change in DNA recognition specificity by RVDs in left (top panel) and right (bottom panel) TALEN arms in the above plasmid samples. Amino acid sequence alignment was performed after translating DNA sequences into amino acids. The pair letters in the sequence represent a RVD (HD, NI, NG, and NN) for each TALE repeat unit in a TALEN arm.
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fig1: Development of a TA cloning- and RE digestion-based method to examine genetic rearrangements of TALE repeat arrays. (a) Work flow chart. A fusion expression cassette bearing both left and right TALEN arms (TALEN L-R) is amplified with forward and reverse PCR primers (FP and RP) designed to amplify the full-length of tandem repeat arrays. The PCR products are inserted into a TA cloning vector. EcoRI digestion of individual clones followed by agarose gel electrophoresis is used to detect the size change of the TALE repeat arrays. The detection of a 1.76 kb DNA fragment indicates that the number of the tandem repeat units remains correct in the TALEN expression cassette. To further classify the genetic rearrangement events, the clones with no obvious size changes are subjected to DNA sequencing. (b) The use of an enzyme mixture allows PCR amplification of single band. Left: Platinum Taq DNA Polymerase High Fidelity. Right: Platinum Taq DNA Polymerase High Fidelity mixed with Elongase (1:2). (c) TA cloning and EcoRI digestion detect no change in the size of the TALE repeat arrays in plasmid vector samples. The band at 3 kb after EcoRI digestion is the linearized cloning vector, while the DNA band at 1.76 kb is the DNA insert bearing a TALE repeat array. “–“ and “+”: Without and with EcoRI. One kb DNA ladder was used as a molecular weight standard. (d) DNA sequencing analysis confirms no change in DNA recognition specificity by RVDs in left (top panel) and right (bottom panel) TALEN arms in the above plasmid samples. Amino acid sequence alignment was performed after translating DNA sequences into amino acids. The pair letters in the sequence represent a RVD (HD, NI, NG, and NN) for each TALE repeat unit in a TALEN arm.

Mentions: To characterize rearrangement events in TALE repeat arrays in a viral vector, we first developed a TA cloning- and RE digestion-based method to examine size change of the arrays (Figure 1a). We used a plasmid vector constructed previously to produce BV-TALEN(L-R), a baculoviral vector with a long repetitive sequence after inserting both left and right TALEN arms into a single expression cassette, to test the method.28 Our initial effort using Platinum Taq DNA Polymerase High Fidelity to amplify the TALE repeat arrays of the plasmid led to a ladder of PCR products differing from the targeted 1.76 kb DNA fragment by multiples of the repeated unit (Figure 1b, left). Generation of such shadow bands is commonly observed when tandem repeat regions are PCR amplified using primers complementary to unique sequences that flank the repeat region and probably due to slipped-strand misalignment by Taq DNA polymerase.34,35 Since Elongase provides a high processivity by editing nascent strands to allow subsequent polymerization by Taq DNA Polymerase, this enzyme mixture was used together with Platinum Taq DNA Polymerase High Fidelity. We also increased the PCR annealing temperature to 72 °C and included 5–8% DMSO as a PCR additive to improve amplification specificity and reduce the slipped-strand extension by Taq DNA polymerase.36


Genetic rearrangements of variable di-residue (RVD)-containing repeat arrays in a baculoviral TALEN system.

Lau CH, Zhu H, Tay JC, Li Z, Tay FC, Chen C, Tan WK, Du S, Sia VK, Phang RZ, Tang SY, Yang C, Chi Z, Liang CC, Ning E, Wang S - Mol Ther Methods Clin Dev (2014)

Development of a TA cloning- and RE digestion-based method to examine genetic rearrangements of TALE repeat arrays. (a) Work flow chart. A fusion expression cassette bearing both left and right TALEN arms (TALEN L-R) is amplified with forward and reverse PCR primers (FP and RP) designed to amplify the full-length of tandem repeat arrays. The PCR products are inserted into a TA cloning vector. EcoRI digestion of individual clones followed by agarose gel electrophoresis is used to detect the size change of the TALE repeat arrays. The detection of a 1.76 kb DNA fragment indicates that the number of the tandem repeat units remains correct in the TALEN expression cassette. To further classify the genetic rearrangement events, the clones with no obvious size changes are subjected to DNA sequencing. (b) The use of an enzyme mixture allows PCR amplification of single band. Left: Platinum Taq DNA Polymerase High Fidelity. Right: Platinum Taq DNA Polymerase High Fidelity mixed with Elongase (1:2). (c) TA cloning and EcoRI digestion detect no change in the size of the TALE repeat arrays in plasmid vector samples. The band at 3 kb after EcoRI digestion is the linearized cloning vector, while the DNA band at 1.76 kb is the DNA insert bearing a TALE repeat array. “–“ and “+”: Without and with EcoRI. One kb DNA ladder was used as a molecular weight standard. (d) DNA sequencing analysis confirms no change in DNA recognition specificity by RVDs in left (top panel) and right (bottom panel) TALEN arms in the above plasmid samples. Amino acid sequence alignment was performed after translating DNA sequences into amino acids. The pair letters in the sequence represent a RVD (HD, NI, NG, and NN) for each TALE repeat unit in a TALEN arm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362386&req=5

fig1: Development of a TA cloning- and RE digestion-based method to examine genetic rearrangements of TALE repeat arrays. (a) Work flow chart. A fusion expression cassette bearing both left and right TALEN arms (TALEN L-R) is amplified with forward and reverse PCR primers (FP and RP) designed to amplify the full-length of tandem repeat arrays. The PCR products are inserted into a TA cloning vector. EcoRI digestion of individual clones followed by agarose gel electrophoresis is used to detect the size change of the TALE repeat arrays. The detection of a 1.76 kb DNA fragment indicates that the number of the tandem repeat units remains correct in the TALEN expression cassette. To further classify the genetic rearrangement events, the clones with no obvious size changes are subjected to DNA sequencing. (b) The use of an enzyme mixture allows PCR amplification of single band. Left: Platinum Taq DNA Polymerase High Fidelity. Right: Platinum Taq DNA Polymerase High Fidelity mixed with Elongase (1:2). (c) TA cloning and EcoRI digestion detect no change in the size of the TALE repeat arrays in plasmid vector samples. The band at 3 kb after EcoRI digestion is the linearized cloning vector, while the DNA band at 1.76 kb is the DNA insert bearing a TALE repeat array. “–“ and “+”: Without and with EcoRI. One kb DNA ladder was used as a molecular weight standard. (d) DNA sequencing analysis confirms no change in DNA recognition specificity by RVDs in left (top panel) and right (bottom panel) TALEN arms in the above plasmid samples. Amino acid sequence alignment was performed after translating DNA sequences into amino acids. The pair letters in the sequence represent a RVD (HD, NI, NG, and NN) for each TALE repeat unit in a TALEN arm.
Mentions: To characterize rearrangement events in TALE repeat arrays in a viral vector, we first developed a TA cloning- and RE digestion-based method to examine size change of the arrays (Figure 1a). We used a plasmid vector constructed previously to produce BV-TALEN(L-R), a baculoviral vector with a long repetitive sequence after inserting both left and right TALEN arms into a single expression cassette, to test the method.28 Our initial effort using Platinum Taq DNA Polymerase High Fidelity to amplify the TALE repeat arrays of the plasmid led to a ladder of PCR products differing from the targeted 1.76 kb DNA fragment by multiples of the repeated unit (Figure 1b, left). Generation of such shadow bands is commonly observed when tandem repeat regions are PCR amplified using primers complementary to unique sequences that flank the repeat region and probably due to slipped-strand misalignment by Taq DNA polymerase.34,35 Since Elongase provides a high processivity by editing nascent strands to allow subsequent polymerization by Taq DNA Polymerase, this enzyme mixture was used together with Platinum Taq DNA Polymerase High Fidelity. We also increased the PCR annealing temperature to 72 °C and included 5–8% DMSO as a PCR additive to improve amplification specificity and reduce the slipped-strand extension by Taq DNA polymerase.36

Bottom Line: The DNA sequence changes in the domains included deletion, addition, substitution, and DNA strand exchange between the left and right TALEN arms.Based on these observations, we have developed a protocol using a low MOI to produce baculoviral vectors expressing TALEN left and right arms separately.Cotransduction of the viruses produced by this optimal protocol provided an improved TALEN cleavage efficiency and enabled effective site-specific transgene integration in human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.

ABSTRACT
Virus-derived gene transfer vectors have been successfully employed to express the transcription activator-like effector nucleases (TALENs) in mammalian cells. Since the DNA-binding domains of TALENs consist of the variable di-residue (RVD)-containing tandem repeat modules and virus genome with repeated sequences is susceptible to genetic recombination, we investigated several factors that might affect TALEN cleavage efficiency of baculoviral vectors. Using a TALEN system designed to target the AAVS1 locus, we observed increased sequence instability of the TALE repeat arrays when a higher multiplicity of infection (MOI) of recombinant viruses was used to produce the baculoviral vectors. We also detected more deleterious mutations in the TALE DNA-binding domains when both left and right TALEN arms were placed into a single expression cassette as compared to the viruses containing one arm only. The DNA sequence changes in the domains included deletion, addition, substitution, and DNA strand exchange between the left and right TALEN arms. Based on these observations, we have developed a protocol using a low MOI to produce baculoviral vectors expressing TALEN left and right arms separately. Cotransduction of the viruses produced by this optimal protocol provided an improved TALEN cleavage efficiency and enabled effective site-specific transgene integration in human cells.

No MeSH data available.


Related in: MedlinePlus