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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: Virus-derived gene transfer vectors have been successfully employed to express the transcription activator-like effector nucleases (TALENs) in mammalian cells.The DNA sequence changes in the domains included deletion, addition, substitution, and DNA strand exchange between the left and right TALEN arms.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

Southern blot analysis to examine the structural integrity of TALE repeat arrays. (a) The probe binding sites used for Southern blot analysis. The probe used was amplified from the FokI cleavage domain in the donor plasmid carrying a fusion expression cassette (TALEN L-R). DNA bearing the TALEN expression cassette was digested with NsiI and XbaI to generate 2.4 kb DNA fragments containing the central TALE repeat array and Fokl cleavage domain. (b) The structural integrity of TALE repeat arrays in the donor plasmids, composite bacmids and baculoviral genome. Genomic DNA of recombinant baculovirus (gBV) was extracted after serial passages (P1, P2, and P3). (c) The influence of MOI used to infect insect cells and the length of tandem repeats in TALEN constructs on structural integrity. As a positive control, a TALEN expression cassette in a donor plasmid vector was digested with BamHI and XbaI to generate a DNA fragment consisting of the FokI cleavage domain only (634 bp).
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fig4: Southern blot analysis to examine the structural integrity of TALE repeat arrays. (a) The probe binding sites used for Southern blot analysis. The probe used was amplified from the FokI cleavage domain in the donor plasmid carrying a fusion expression cassette (TALEN L-R). DNA bearing the TALEN expression cassette was digested with NsiI and XbaI to generate 2.4 kb DNA fragments containing the central TALE repeat array and Fokl cleavage domain. (b) The structural integrity of TALE repeat arrays in the donor plasmids, composite bacmids and baculoviral genome. Genomic DNA of recombinant baculovirus (gBV) was extracted after serial passages (P1, P2, and P3). (c) The influence of MOI used to infect insect cells and the length of tandem repeats in TALEN constructs on structural integrity. As a positive control, a TALEN expression cassette in a donor plasmid vector was digested with BamHI and XbaI to generate a DNA fragment consisting of the FokI cleavage domain only (634 bp).

Mentions: We further performed Southern blot analysis to examine the structural integrity of the TALE repeat arrays in the donor plasmids, composite bacmids, and baculoviral genome. Plasmid, bacmid, and viral genomic DNA were digested with NsiI and XbaI before agarose gel electrophoresis. Instead of trying to differentiate the extent of mutations between left and right TALEN arms, we used these two REs to determine the overall structural integrity of the two TALEN arms in the expression cassette L-R. The Southern blot probe was designed to bind to the FokI region, which is located downstream of a TALE DNA binding domain (Figure 4a). The detection of a single 2.4 kb DNA fragment bearing the FokI and TALE DNA binding domains in the donor plasmids and composite bacmids demonstrated the TALE repeat arrays remain unchanged in these samples (Figure 4b). Upon transfection of insect cells with the composite bacmids to generate infectious recombinant baculovirus, “shadow” bands were observed in Southern blots for the early passage (P1) baculoviruses, indicating possible genetic rearrangements in the TALE repeat arrays. These events appeared to increase from P1 to P3, possibly due to the accumulation of genetic rearrangements over virus passaging. We then examined the genomic DNA of late passage (P3) baculoviruses generated with various MOIs of P2 viral stocks, from 0.005, 0.025, 0.05, 0.10 to 0.20 pfu per cell, and observed a ‘ladder’ of bands starting at ~600 bp and every 100 bp up to ~2,300 bp (Figure 4c). With an increase in MOI of P2 viral stock, P3 BV-TALEN(L-R) bands with intact DNA size (2.4 kb) became less noticeable, indicating the disappearance of functional TALENs. Thus, our Southern blot analysis, although unable to provide quantitative evaluation, shows again that low multiplicity of infection to produce baculoviral vectors with one TALEN arm only will be favorable to generation of functional viral TALEN vectors.


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)

Southern blot analysis to examine the structural integrity of TALE repeat arrays. (a) The probe binding sites used for Southern blot analysis. The probe used was amplified from the FokI cleavage domain in the donor plasmid carrying a fusion expression cassette (TALEN L-R). DNA bearing the TALEN expression cassette was digested with NsiI and XbaI to generate 2.4 kb DNA fragments containing the central TALE repeat array and Fokl cleavage domain. (b) The structural integrity of TALE repeat arrays in the donor plasmids, composite bacmids and baculoviral genome. Genomic DNA of recombinant baculovirus (gBV) was extracted after serial passages (P1, P2, and P3). (c) The influence of MOI used to infect insect cells and the length of tandem repeats in TALEN constructs on structural integrity. As a positive control, a TALEN expression cassette in a donor plasmid vector was digested with BamHI and XbaI to generate a DNA fragment consisting of the FokI cleavage domain only (634 bp).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Southern blot analysis to examine the structural integrity of TALE repeat arrays. (a) The probe binding sites used for Southern blot analysis. The probe used was amplified from the FokI cleavage domain in the donor plasmid carrying a fusion expression cassette (TALEN L-R). DNA bearing the TALEN expression cassette was digested with NsiI and XbaI to generate 2.4 kb DNA fragments containing the central TALE repeat array and Fokl cleavage domain. (b) The structural integrity of TALE repeat arrays in the donor plasmids, composite bacmids and baculoviral genome. Genomic DNA of recombinant baculovirus (gBV) was extracted after serial passages (P1, P2, and P3). (c) The influence of MOI used to infect insect cells and the length of tandem repeats in TALEN constructs on structural integrity. As a positive control, a TALEN expression cassette in a donor plasmid vector was digested with BamHI and XbaI to generate a DNA fragment consisting of the FokI cleavage domain only (634 bp).
Mentions: We further performed Southern blot analysis to examine the structural integrity of the TALE repeat arrays in the donor plasmids, composite bacmids, and baculoviral genome. Plasmid, bacmid, and viral genomic DNA were digested with NsiI and XbaI before agarose gel electrophoresis. Instead of trying to differentiate the extent of mutations between left and right TALEN arms, we used these two REs to determine the overall structural integrity of the two TALEN arms in the expression cassette L-R. The Southern blot probe was designed to bind to the FokI region, which is located downstream of a TALE DNA binding domain (Figure 4a). The detection of a single 2.4 kb DNA fragment bearing the FokI and TALE DNA binding domains in the donor plasmids and composite bacmids demonstrated the TALE repeat arrays remain unchanged in these samples (Figure 4b). Upon transfection of insect cells with the composite bacmids to generate infectious recombinant baculovirus, “shadow” bands were observed in Southern blots for the early passage (P1) baculoviruses, indicating possible genetic rearrangements in the TALE repeat arrays. These events appeared to increase from P1 to P3, possibly due to the accumulation of genetic rearrangements over virus passaging. We then examined the genomic DNA of late passage (P3) baculoviruses generated with various MOIs of P2 viral stocks, from 0.005, 0.025, 0.05, 0.10 to 0.20 pfu per cell, and observed a ‘ladder’ of bands starting at ~600 bp and every 100 bp up to ~2,300 bp (Figure 4c). With an increase in MOI of P2 viral stock, P3 BV-TALEN(L-R) bands with intact DNA size (2.4 kb) became less noticeable, indicating the disappearance of functional TALENs. Thus, our Southern blot analysis, although unable to provide quantitative evaluation, shows again that low multiplicity of infection to produce baculoviral vectors with one TALEN arm only will be favorable to generation of functional viral TALEN vectors.

Bottom Line: Virus-derived gene transfer vectors have been successfully employed to express the transcription activator-like effector nucleases (TALENs) in mammalian cells.The DNA sequence changes in the domains included deletion, addition, substitution, and DNA strand exchange between the left and right TALEN arms.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