File S1 - Analysis of the Role of Homology Arms in Gene-Targeting Vectors in Human Cells

Figures S1-S9. Figure S1. Schematic representation of repetitive DNA sequences present in the HPRT vectors used in this study. The location and length (bp) of each SINE/LINE fragment is based on the UCSC Genome Browser Database: Update 2006 (Nucleic Acids Res. 34:D590–D598, 2006). Figure S2. Impact of siRNA-mediated knockdown of DNA ligase I or IIIα on integration frequency. (A) The nucleotide sequence of LIG1 and LIG3 siRNA. These siRNAs were designed as reported previously (Nucleic Acids Res. 36: 3297–3310, 2008). (B) Western blot analysis for DNA ligase I and IIIα in siRNA-transfected Nalm-6 wild-type and LIG4-null cells. M, mock-transfected. (C, D) Integration frequency in wild-type and LIG4-null cells treated with LIG1 siRNA (C) or LIG3 siRNA (D). A non-targeting vector (pβactin-His; Nucleic Acids Res. 36: 6333–6342, 2008) was used for transfection. The integration frequency in mock-transfected wild-type cells was taken as 1, and the relative integration frequency was calculated. Figure S3. Structural features of gene-targeting vectors used for the analysis of integration frequency. (A) Fundamental structure of targeting vectors. In all the vectors, 5' and 3' arms flank the drug-resistance gene cassette (Puror), which is placed in the forward direction. (B) Structural features of the fourteen gene-targeting vectors used. Shown are the lengths of 5' and 3' arms and SINE/LINE sequences within each arm and the integration frequency. The length of SINE/LINE is based on the UCSC Genome Browser Database: Update 2006 (Nucleic Acids Res. 34:D590–D598, 2006). Figure S4. Integration frequency of targeting vector as a function of the length of repetitive DNA sequences. Integration frequencies of pHPRT8.9-Puro(+), pHPRT2.2-Puro(+), and twelve other gene-targeting vectors are shown as a function of the total length of SINE sequence (A), 5’-arm SINE length (B), 3’-arm SINE length (C), the total length of LINE sequence (D), 5’-arm LINE length (E), and 3’-arm LINE length (F). See also Figure 5. Figure S5. Correlation between the integration frequency and repetitive DNA sequences. (A) Integration frequencies of pHPRT8.9-Puro(+), pHPRT2.2-Puro(+), and twelve other gene-targeting vectors as a function of the total length of SINE/LINE sequences. Note that this graph is the same as Figure 5D. (B) Same as (A), except that the three vectors have been omitted from the data set. Note that the redrawn fitted curve reveals a stronger correlation between the total SINE/LINE length and the integration frequency. See text and Figure 5 for details. Figure S6. Gene-targeting efficiency is little affected by the length of homology arms. Targeting efficiencies are shown as a function of the total length of homology arms of the targeting vector. The twelve non-HPRT gene-targeting vectors were used for the analysis (see Figure S3B in File S1). Figure S7. PCR primers used to amplify the homology arms of pHPRT2.2-Puro vectors. The restriction sites used to construct the arm-deleted vectors are shown in red (SacI) or blue (SalI). Figure S8. PCR primers used to amplify the homology arms of non-HPRT targeting vectors. Red denotes attB sequences. Figure S9. Schematic representation of construction of imperfect pHPRT8.9-Puro vectors lacking the 3' arm (A) or 5' arm (B).
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