Different Frequency of Gene Targeting Events by the RNA-DNA Oligonucleotide Among Epithelial Cells Evelyn Santana, Adam E. Peritz, Subramanian Iyer,

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Presentation transcript:

Different Frequency of Gene Targeting Events by the RNA-DNA Oligonucleotide Among Epithelial Cells Evelyn Santana, Adam E. Peritz, Subramanian Iyer, Jouni Uitto, Kyonggeun Yoon Journal of Investigative Dermatology Volume 111, Issue 6, Pages 1172-1177 (December 1998) DOI: 10.1046/j.1523-1747.1998.00403.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Sequences of the RNA-DNA oligonucleotide (AP2 and SC2) and the targeted sequences in alkaline phosphatase and β-globin genes. The site of change is indicated by the asterisk in the sequence. DNA residues are capitalized and 2′-O-methyl RNA residues are in lower case. Journal of Investigative Dermatology 1998 111, 1172-1177DOI: (10.1046/j.1523-1747.1998.00403.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Cellular uptake of RNA-DNA oligonucleotide in human primary keratinocytes, HaCaT cells, and HeLa cells. (A) Nuclear uptake; (B) cytoplasmic uptake. Uptake is expressed in pmol of oligonucleotide per 100,000 cells. Journal of Investigative Dermatology 1998 111, 1172-1177DOI: (10.1046/j.1523-1747.1998.00403.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Stability of the RNA-DNA oligonucleotide. HeLa cells were transfected with the32P end-labeled oligonucleotide as described in theMaterials and Methods. Oligonucleotide was extracted from the cytoplasm and the nucleus at various time intervals, i.e., 0, 6, 24, and 48 h after transfection. M shows32P end-labeled 10 bp marker and thearrow indicates an intact full-length oligonucleotide. A full-length 68-mer RNA-DNA oligonucleotide migrates slightly faster than the position 68, because of a compact double-stranded structure of RNA-DNA oligonucleotide that is not completely denatured in the 7 M urea. The size of the degraded product is between 50 and 60 base, indicating a cleavage site at the junction between 2′-O-methyl RNA and DNA at the position 54. Journal of Investigative Dermatology 1998 111, 1172-1177DOI: (10.1046/j.1523-1747.1998.00403.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Uptake of the fluorescein-conjugated RNA–DNA oligonucleotide. Confocal micrograph of human primary keratinocytes (A), HaCaT cells (B), and HeLa cells (C) transfected with fluorescein-conjugated oligonucleotide at 160 nM.Scale bar: 8 μm. Journal of Investigative Dermatology 1998 111, 1172-1177DOI: (10.1046/j.1523-1747.1998.00403.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 RFLP analysis of gene conversion by AP2. The extent of gene conversion event by AP2 in human primary keratinocytes (A), HaCaT cells (B), and HeLa cells (C). The wild-type sequence of the bone/liver/kidney alkaline phosphatase (GCCCACTCGGC) is cleaved by the restriction enzymeBglI resulting in 114 and 80 bp fragments, whereas the mutated sequence (ACCCACTCGGC) is not cleaved and results in a 194 bp fragment indicated by anarrow.Lane 1, 100 bp marker;lanes 2, 4, 6, and 8 contain PCR products from cells treated with 0, 20, 110, and 160 nM AP2, respectively.Lanes 3, 5, 7, and 9 showBglI digestion of the PCR products inlanes 2, 4, 6, and 8, respectively. Part (D) depicts a direct sequencing of the 194 bp PCR product. A base change (C→T) in the complementary strand was observed, indicating a G→A change in the coding strand at a position indicated by anarrow. Journal of Investigative Dermatology 1998 111, 1172-1177DOI: (10.1046/j.1523-1747.1998.00403.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 RFLP analysis of gene conversion by SC2. The extent of gene conversion event by SC2 in human primary keratinocytes (A), HaCaT cells (B), and HeLa cells (C). The wild-type sequence of β-globin (CCTGAGG) is cleaved byDdeI, resulting in 189 and 108 bp fragments, whereas the mutated sequence (CCTGTGG) results in an 297 bp fragment indicated by anarrow.Lane 1, 100 bp marker;lanes 2, 4, 6, and 8 contain PCR products from cells treated with 0, 20, 110, and 160 nM SC2, respectively.Lanes 3, 5, 7, and 9 showDdeI digestion of the PCR products inlanes 2, 4, 6, and 8, respectively. Journal of Investigative Dermatology 1998 111, 1172-1177DOI: (10.1046/j.1523-1747.1998.00403.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions