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Optimization and analysis of single-...

Murphy, Brian.

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Optimization and analysis of single-stranded oligonucleotide directed gene targeting in mouse embryonic stem cells.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Optimization and analysis of single-stranded oligonucleotide directed gene targeting in mouse embryonic stem cells./
作者:	Murphy, Brian.
面頁冊數:	106 p.
附註:	Adviser: Scott Diamond.
Contained By:	Dissertation Abstracts International68-04B.
標題:	Biology, Molecular. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3260955

Optimization and analysis of single-stranded oligonucleotide directed gene targeting in mouse embryonic stem cells.
Murphy, Brian.

Optimization and analysis of single-stranded oligonucleotide directed gene targeting in mouse embryonic stem cells. - 106 p.

Adviser: Scott Diamond.

Thesis (Ph.D.)--University of Pennsylvania, 2007.

Single stranded oligodeoxynucleotide (ssODN) gene targeting may facilitate animal model creation and gene repair therapy. Lipofection of ssODN can introduce point mutations into target genes. However, typical efficiencies in mouse embryonic stem cells (mES) are <10-4, leaving corrections too rare to effectively identify. We developed mES lines with an integrated mutant neomycin resistance gene (Tyr22Ter). After targeting with ssODN, repaired cells survive selection in G418. Corrected cells have normal phenotypes and have been injected into blastocysts to create chimeric mice. Germline transmission has not been observed to date.Subjects--Topical Terms:

1017719
Biology, Molecular.

Optimization and analysis of single-stranded oligonucleotide directed gene targeting in mouse embryonic stem cells.
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245 1 0 $a Optimization and analysis of single-stranded oligonucleotide directed gene targeting in mouse embryonic stem cells.
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502 $a Thesis (Ph.D.)--University of Pennsylvania, 2007.
520 $a Single stranded oligodeoxynucleotide (ssODN) gene targeting may facilitate animal model creation and gene repair therapy. Lipofection of ssODN can introduce point mutations into target genes. However, typical efficiencies in mouse embryonic stem cells (mES) are <10-4, leaving corrections too rare to effectively identify. We developed mES lines with an integrated mutant neomycin resistance gene (Tyr22Ter). After targeting with ssODN, repaired cells survive selection in G418. Corrected cells have normal phenotypes and have been injected into blastocysts to create chimeric mice. Germline transmission has not been observed to date.
520 $a Correction efficiencies varied with different transfection methods, clonal lines, and ssODN designs, ranging from 1 to 100 corrections per million cells plated. Uptake studies using cell sorting of Cy5-labelled ssODN showed 47% of the corrections concentrated in the best transfected 22% of cells. Four different basepair mismatches were tested and results show that the base-specificity of the mismatch is critical. An ssODN with a G:A mismatch is approximately 6 times more efficient than an otherwise identical sequence with a G:G mismatch. Dual mismatch ssODN with silent mutations 9 bases downstream also showed mismatch preferences. There, the A:G mismatch was most efficient.
520 $a Although correction efficiencies can be highly variable, a model is proposed to separate variables and assist in data analysis. Data is shown to be Poisson distributed. Other technologies such as zinc finger nucleases, RNAi, methylated oligonucleotides, and high throughput screening were examined, but have not boosted efficiencies to the required threshold. The neomycin selectable system is a convenient assay for the testing of new parameters in mES, and these lines may facilitate development of improved ssODN targeting technologies for either animal production or ex vivo gene therapy.
590 $a School code: 0175.
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650 4 $a Engineering, Chemical. $3 1018531
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