Gene reporter is a valuable in vitro tool that facilitates live monitoring and tracking a cell type of interest. Lineage reporters are either created using minimal lineage-specific promoter-driven reporter systems or via knock-in of the reporter into the endogenous promoter region. Choice of the reporter is critical based on intended application.

1. Email: info@creative-biogene.com
Website: http://www.creative-biogene.com
Reporter Stem Cells Generation
Creative
Biogene

2. Introduction
Gene Reporter Pluripotent Lines
Methods for Reporter Cells Generation
Potential Uses of Reporter Stem Cells
About Creative Biogene

3. Human pluripotent stem cells (PSCs) such as induced pluripotent stem cells (iPSC) and embryonic stem cells
(hESC) hold great promise in the regenerative medicine. PSCs and their ability to form any cell type in the
human body has allowed researchers to use them as research tools for applications in basic developmental
research, drug screening and potential therapeutic implementations.
An emerging need for stem cell biology is efficient and
homogeneous differentiation of stem cells into mature, specialized,
functional cells. Reporter stem cell lines are valuable models which
enable noninvasive, live monitoring of marker onset and expression
in a cell-specific manner. Some methods have been used to derive
such cell lines based on lineage promoter-driven reporter expression.
A more regulated expression achieved with a reporter knock-in into
the endogenous promoter loci was less utilized due to the associated
technical difficulty. While new advances in genome-editing
technologies have lowered these barriers for creating knock-in
reporter lines.
Introduction

4. Gene Reporter Pluripotent Lines as Cell Models
Gene reporter is a valuable in vitro tool that facilitates live monitoring and tracking a cell type of interest. Lineage
reporters are either created using minimal lineage-specific promoter-driven reporter systems or via knock-in of the
reporter into the endogenous promoter region. Choice of the reporter is critical based on intended application. Although
there is a wide choice of reporters that can be used, each system offers its own advantages and disadvantages.
Promoter
Method Gene mMarker Advantages Disadvantages
Gene tags: bLac,bGal, AP
Enzymatic methods and hence
signal can be amplified
Cells need to be fixed for
visualization
Fluorescent proteins:
GFP/CFP/YFP, DsRED
Enables visualization of cells
High-level expression is required
to be visualized over
autofluorescence
Bioluminescent
marker: luciferase
Detection of sensitivity and use in
biochemical assays
Requires cofactors/substrates
and specialized equipment
Drug selectable markers:
Hyg,Neo, Puro,BSD
Sensitive even at low expression
levels
Poor survival at low density,
does not allow visualization
Suicide genes: HSV-
TK,diphtheria toxin-A
Allows enrichment or elimination
of desired cells
Poor survival at low density,
does not allow visualization
Exogenous
lineage promoter or
endogenous
promoter Loci
Reporter

5. Methods for Reporter Cells Generation
 Minimal Promoter-Driven Reporter Expression
Exogenous lineage-specific promoter and the regulatory fragments can be cloned upstream of a reporter gene to
generate cell-specific promoter reporters. In most instances, DNA sequences upstream of the gene-encoding regions
containing cis-acting elements are designated as the promoter region; the represents a very restrictive representation
of the regulatory elements as in some cases gene regulation is known to be complex, requiring the combination of
distal and proximal elements.
Therefore, the primary concern about the use of promoter- reporter systems is that the reporter may fail to faithfully
recapitulate the activity of the endogenous target gene. And it is essential to carry out extensive validation of the
promoter-reporter constructs to confirm context-specific expression of the reporter. Expression level is also much
dependent on how the construct is delivered for stable expression into host cells. The promoter-reporter construct can
either be maintained episomally without integration, or integrated either randomly or site-specifically into the host
genome.

6. Methods for generation of lineage reporter lines using lineage promoter-driven reporter constructs as follows.
Nonintegrational methods using episomal vectors prevent genomic integration of the transgene, whereas integration
methods result in either random insertion or site-specific integration of the transgene into the host genome.

7. Several viral and DNA-based methods facilitate the integration of delivered
DNA constructs into the host genome, either in a random manner or to
specific loci in the genome. Random integration of the promoter-reporter
construct can be achieved via naked DNA delivery or as lentiviral particles.
In both cases, high copy numbers of the construct can be achieved with
efficient delivery into cells.
Epstein–Barr virus (EBV)-based episomal vectors have been successfully
used to stably express the gene of interest in multiple types of cells
without integrating into the host genome. This system offers an appealing
alternative because it is relatively free from chromosomal effects
associated with genomic integration methods and has been used to create
pluripotent reporter lines with Pou5F1 (Oct4) promoter.
Nonintegrational Methods
Integrational Methods

8. Methods for Reporter Cells Generation
 Reporter Knock-In into Endogenous Promoter Site
Reporter genes can be inserted into specific genomic sites of interest via homologous recombination. The
targeting construct composed of a core region carrying the reporter cassette is flanked by homology arms that
recombine with the target genomic loci, resulting in site-specific insertion of the reporter. This process is fairly
inefficient, generally occurring at a rate of one in a million cells that can be significantly enhanced in the presence
of double-strand breaks.
New gene-targeting technologies aim to precisely cleave genomic loci to facilitate insertion or deletion of genes
at the specific cleavage site. Several reporter lines in ESC and iPSC have been generated using these methods.

9. (A) Traditional methods relied on homologous recombination between the target genomic site at the endogenous
promoter locus and homology arm flanking the reporter. (B) Meganucleases that use engineered versions of naturally
occurring restriction enzymes with extended recognition sequences comprise both the DNA recognition and cleavage
on a single domain. The newer systems (C) ZFN, (D) TALENS, and (E) CRISPR rely on engineered molecular scissors that
precisely create double-strand breaks at specific genomic sites.

10. CRISPR/Cas9 method
The most recent advent in genome editing has been the development of clustered regularly interspaced palindromic repeat
(CRISPR)-guided Cas9 nuclease-mediated cleavage. There is a major aspect of CRISPR/Cas9 that makes it unique. The Cas9
nuclease remains independent of the guide sequence until the two are introduced upon performing an experiment. Having this
flexibility means that targeting multiple sites is relatively simple; as the CRISPR/Cas9 method only requires the synthesis of only a
new guide RNA, instead of the production of a new target-specific nuclease from scratch. The biggest advantage of CRISPR-Cas9 is
the specificity, efficiency, and ease with which the system can be utilized to target multiple sites, individually or together.

11. Potential Uses of Reporter Stem Cells
Reporters can either be expressed in the pluripotent state and turned off with differentiation or vice versa, where the
reporter expression is off to begin with and is turned on with differentiation. Reporter lines enable isolation of a
homogeneous population of cells that is critical for characterization and for use in downstream applications. In addition, a
visual reporter offers the opportunity of tracking cell fate progression in live cells.

12. Based on years of experience and in-depth investigation,
scientists at Creative Biogene can target specific DNA
sequences and induce a double-stranded break, taking
advantage of recombination to create synthetic genetic
genome of a host.
An expression cassette containing reporter and
resistance genes is knocked into the safe-harbor site or
any other sites in host cells. Gene insertion at a safe-
harbor site allows stable gene expression without any
adverse effects on the fitness of the engineered cells.
About Creative Biogene