Abstract
Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, "Small Fragment Homologous Replacement (SFHR)", has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely last any measurable CFTR-dependent chloride efflux The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.
| Original language | English |
|---|---|
| Pages (from-to) | 2989-2999 |
| Number of pages | 11 |
| Journal | Frontiers in Bioscience |
| Volume | 13 |
| Issue number | 8 |
| DOIs | |
| Publication status | Published - 2008 |
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Keywords
- CFTR
- Embryonic stem cells
- Homologous replacement
- Real-time PCR
- SFHR
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Cell Biology
- Medicine(all)
Cite this
CFTR gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR). / Sangiuolo, Federica; Scaldaferri, Maria Lucia; Filareto, Antonio; Spitalieri, Paola; Guerra, Lorenzo; Favia, Maria; Caroppo, Rosa; Mango, Ruggiero; Bruscia, Emanuela; Gruenert, Dieter C.; Casavola, Valeria; De Felici, Massimo; Novelli, Giuseppe.
In: Frontiers in Bioscience, Vol. 13, No. 8, 2008, p. 2989-2999.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - CFTR gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR)
AU - Sangiuolo, Federica
AU - Scaldaferri, Maria Lucia
AU - Filareto, Antonio
AU - Spitalieri, Paola
AU - Guerra, Lorenzo
AU - Favia, Maria
AU - Caroppo, Rosa
AU - Mango, Ruggiero
AU - Bruscia, Emanuela
AU - Gruenert, Dieter C.
AU - Casavola, Valeria
AU - De Felici, Massimo
AU - Novelli, Giuseppe
PY - 2008
Y1 - 2008
N2 - Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, "Small Fragment Homologous Replacement (SFHR)", has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely last any measurable CFTR-dependent chloride efflux The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.
AB - Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, "Small Fragment Homologous Replacement (SFHR)", has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely last any measurable CFTR-dependent chloride efflux The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.
KW - CFTR
KW - Embryonic stem cells
KW - Homologous replacement
KW - Real-time PCR
KW - SFHR
UR - http://www.scopus.com/inward/record.url?scp=38449096253&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=38449096253&partnerID=8YFLogxK
U2 - 10.2741/2904
DO - 10.2741/2904
M3 - Article
C2 - 17981772
AN - SCOPUS:38449096253
VL - 13
SP - 2989
EP - 2999
JO - Frontiers in Bioscience - Landmark
JF - Frontiers in Bioscience - Landmark
SN - 1093-9946
IS - 8
ER -