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Transient gene delivery for functional enrichment of differentiating embryonic stem cells

Descriptive

TypeOfResource
Text
TitleInfo (ID = T-1)
Title
Transient gene delivery for functional enrichment of differentiating embryonic stem cells
Identifier
ETD_1391
Identifier (type = hdl)
http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000051077
Language
LanguageTerm (authority = ISO639-2); (type = code)
eng
Genre (authority = marcgt)
theses
Subject (ID = SBJ-1); (authority = RUETD)
Topic
Biomedical Engineering
Subject (ID = SBJ-1); (authority = ETD-LCSH)
Topic
Genetic transcription
Subject (ID = SBJ-1); (authority = ETD-LCSH)
Topic
Gene expression
Abstract
There is a critical need for new sources of hepatocytes, both clinically to provide support for patients with liver failure and in drug discovery for toxicity, metabolic and pharmacokinetic screening of new drug entities. One major challenge in the field of differentiating embryonic stem (ES) cells is the limitation to selectively purify and enrich these cells from a heterogeneous population. We developed a transient gene delivery system that uses fluorescent gene reporters for purification of the cells. Following a transient transfection, the cells were purified through a fluorescence-activated cell sorter (FACS), re-plated in secondary culture and subsequent phenotypic analysis performed. We engineered two non-viral plasmid reporters, the first driven by the mouse albumin enhancer/promoter and the second by the mouse cytochrome P450 7A1 (Cyp7A1) promoter. We optimized the transfection of these genes into spontaneously differentiated ES cells and sorted independent fractions positive for each reporter 17 days after inducing differentiation. We found that cells sorted based on the Cyp7A1 promoter showed significant enrichment in terms of albumin secretion, urea secretion and cytochrome P450 1A2 detoxification activity as compared to enrichment garnered by the albumin promoter-based cell sort. In a second study, we explored improving the efficiency of a transient gene delivery system to differentiating ES cells by serum starving the cells for three days. We found that under serum starvation, expression of a constitutively-controlled plasmid increases from ~50% to ~83% of the population. When probed with the Cyp7A1 liver-specific reporter vector, the expression increases from ~1.4% to ~3.7% of the population. These trends were assessed using a Cy3-tagged oligonucleotide, which enabled rapid quantification of DNA uptake and was a valid predictor of ultimate cell transfection efficiency. These results suggest that modifications in media components prior to transfection of cells can have a profound effect on improving non-viral gene delivery. Efficiently genetically engineering cellular systems while circumventing the need to induce permanent gene changes will be critical for the generation of clinically-acceptable cellular material in the future.
PhysicalDescription
Form (authority = gmd)
electronic resource
Extent
xii, 92 p. : ill.
InternetMediaType
application/pdf
InternetMediaType
text/xml
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references (p. 82-90)
Note (type = statement of responsibility)
by Eric J. Wallenstein
Name (ID = NAME-1); (type = personal)
NamePart (type = family)
Wallenstein
NamePart (type = given)
Eric J.
Role
RoleTerm (authority = RULIB); (type = )
author
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Eric J. Wallenstein
Name (ID = NAME-2); (type = personal)
NamePart (type = family)
Yarmush
NamePart (type = given)
Martin
Role
RoleTerm (authority = RULIB); (type = )
chair
Affiliation
Advisory Committee
DisplayForm
Martin l Yarmush
Name (ID = NAME-3); (type = personal)
NamePart (type = family)
Roth
NamePart (type = given)
Charles
Role
RoleTerm (authority = RULIB); (type = )
internal member
Affiliation
Advisory Committee
DisplayForm
Charles M Roth
Name (ID = NAME-4); (type = personal)
NamePart (type = family)
Kim
NamePart (type = given)
Sobin
Role
RoleTerm (authority = RULIB); (type = )
internal member
Affiliation
Advisory Committee
DisplayForm
Sobin Kim
Name (ID = NAME-5); (type = personal)
NamePart (type = family)
Schloss
NamePart (type = given)
Rene
Role
RoleTerm (authority = RULIB); (type = )
internal member
Affiliation
Advisory Committee
DisplayForm
Rene S Schloss
Name (ID = NAME-6); (type = personal)
NamePart (type = family)
Berthiaume
NamePart (type = given)
Fran´┐Żois
Role
RoleTerm (authority = RULIB)
outside member
Affiliation
Advisory Committee
DisplayForm
Fran´┐Żois Berthiaume
Name (ID = NAME-1); (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB); (type = )
degree grantor
Name (ID = NAME-2); (type = corporate)
NamePart
Graduate School - New Brunswick
Role
RoleTerm (authority = RULIB); (type = )
school
OriginInfo
DateCreated (point = ); (qualifier = exact)
2009
DateOther (qualifier = exact); (type = degree)
2009-01
Place
PlaceTerm (type = code)
xx
Location
PhysicalLocation (authority = marcorg)
NjNbRU
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
RelatedItem (type = host)
TitleInfo
Title
Graduate School - New Brunswick Electronic Theses and Dissertations
Identifier (type = local)
rucore19991600001
Identifier (type = doi)
doi:10.7282/T3ST7Q2R
Genre (authority = ExL-Esploro)
ETD doctoral
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Rights

RightsDeclaration (AUTHORITY = GS); (ID = rulibRdec0006)
The author owns the copyright to this work.
Copyright
Status
Copyright protected
Availability
Status
Open
RightsEvent (AUTHORITY = rulib); (ID = 1)
Type
Permission or license
Detail
Non-exclusive ETD license
AssociatedObject (AUTHORITY = rulib); (ID = 1)
Type
License
Name
Author Agreement License
Detail
I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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Technical

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ETD
MimeType (TYPE = file)
application/pdf
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application/x-tar
FileSize (UNIT = bytes)
4096000
Checksum (METHOD = SHA1)
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