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Methods Mol Biol. Author manuscript; available in PMC 2015 May 04.
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Published in final edited form as:

Methods Mol Biol. 2014 ; 1194: 289–312. doi:10.1007/978-1-4939-1215-5_16.

Generation of Transgenic Mouse Fluorescent Reporter Lines for

Studying Hematopoietic Development
Andrei M. Vacarua,d, Joseph Vitalea,d, Johnathan Nievesa,d, and Margaret H. Barona,b,c,d,f,*
aDepartment of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
bDepartment of Developmental and Regenerative Biology, Icahn School of Medicine at Mount
Sinai, New York, NY 10029, USA
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cDepartment of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY
10029, USA
dThe Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
eTheBlack Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY
10029, USA
fGraduateSchool of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York,
NY 10029, USA

Abstract
During the development of the hematopoietic system, at least 8 distinct lineages are generated in
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the mouse embryo. Transgenic mice expressing fluorescent proteins at various points in the
hematopoietic hierarchy, from hematopoietic stem cell to multipotent progenitors to each of the
final differentiated cell types, have provided valuable tools for tagging, tracking, and isolating
these cells. In this chapter, we discuss general considerations in designing a transgene, survey
available fluorescent probes, and methods for confirming and analyzing transgene expression in
the hematopoietic systems of the embryo, fetus, and postnatal/adult animal.

Keywords
hematopoiesis; transgenic mice; knock-in; green fluorescent protein; fluorescent reporter

1. Introduction
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Since the discovery and cloning of the first fluorescent protein (FP), wild type green
fluorescent protein (wtGFP) from the bioluminescent jellyfish Aequorea victoria (1) and the
subsequent creation of spectral variants (2–4), FPs have become indispensable for imaging
cellular differentiation and function at high resolution and in real time (reviewed in refs. 3,
4). Gene-specific regulatory elements can be used to drive targeted expression of FP
reporters, with spatial- and/or temporal specificity, in virtually any cell type of a transgenic

*
Corresponding author: Margaret H. Baron, MD PhD, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box
1079, New York, NY 10029-6574, Phone: (212) 241-0825, FAX: (212) 849-2442, margaret.baron@mssm.edu.
 Vacaru et al. Page 2

animal. The hematopoietic system has benefitted enormously from this approach, which
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made it possible to explore the emergence, expansion, migration and differentiation of

progenitors for the erythroid, myeloid, and lymphoid lineages. It is now possible to label and
track the development of distinct hematopoietic cell types in vivo and to isolate these cells
directly, using fluorescence activated cell sorting (FACS). In this chapter, we discuss the
steps involved in the generation and analysis of transgenic lines in which fluorescent
reporters are expressed in hematopoietic lineages of the mouse, the most genetically
tractable model for mammalian development.

1.1 Ontogeny of the mouse hematopoietic system

Hematopoiesis is a precisely orchestrated, stepwise process that leads to the formation of all
lineages of the blood (5). Primitive erythroid cells (EryP) are the first hematopoietic lineage
to detected in the mouse embryo (reviewed in ref. 6). They are generated late in gastrulation,
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in the in the blood islands of the yolk sac (YS), along with macrophages and
megakaryocytes (7). The first definitive hematopoietic cells, comprising erythroid,
megakaryocytic and myeloid lineages, also arise in the YS, shortly after the appearance of
EryP (reviewed in refs. 6, 7). Hematopoietic stem cells (HSCs) form in the aorta-gonad-
mesonephros (AGM) region of the embryo, in the large arteries and placenta, and, very
likely, in the YS(for a review, see ref. 6). They do not differentiate in these sites but instead
seed the fetal liver (FL), where they expand and produce progenitors that give rise to
definitive erythro-myeloid and lymphoid lineages. Late in gestation, HCSs migrate from the
fetal liver to the bone marrow, which becomes the main blood production center in the
postnatal animal (8). The general hierarchy of hematopoietic development is shown as a
"snapshot" in ref. (9).

1.2 General Considerations in Designing a Transgene

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Transgenic mouse lines expressing fluorescent proteins (FPs) are invaluable tools for
studying the development of the hematopoietic system. Careful design is necessary to
achieve the desired expression of the reporter protein. The main components of a fluorescent
reporter transgene are the promoter (and, usually, other upstream regulatory sequences),
sequences encoding the fluorescent protein, and splice/polyadenylation signals (Figure 1). A
cartoon outlining the most commonly used approach for creation of a transgenic mouse line
is presented in Figure 2.

1.2.1 Regulatory Elements—The promoter is the region of a gene from which mRNA
transcription is initiated and is essential for controlling both the spatial and temporal
expression of a transgene. A number of hematopoietic specific promoters have been used
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successfully to drive expression of fluorescent reporters in different hematopoietic lineages

(see Table 1). The transgene construct should include a translational start codon (ATG), a
Kozak sequence either upstream from or coupled to the start codon (10), and a translational
stop codon (Figure 1). Posttranscriptional regulatory elements may be included to enhance
mRNA stability (e.g. see ref. 11).

Additional regulatory elements should accompany the promoter to drive the desired
transgene expression pattern. The most commonly used regulatory elements are enhancers

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or other upstream regulatory elements (12), an intron, which provides splice donor and
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acceptor sites, and a polyadenylation signal (Figure 1). The intron may be taken from the
same gene as the promoter. Low transgene expression may be significantly increased
through the use of a generic intron (13).

It is well documented that prokaryotic sequences in the vector perturb the frequency and
extent of transgene expression (14–16). Therefore, restriction enzyme sites flanking the
transgene should be included to allow removal of the vector backbone before microinjection.

Following microinjection into the male pronucleus of a fertilized egg (Figure 2), the
transgene is inserted randomly, and often in multiple copies, into the genome (16). The
neighboring chromatin may influence expression of the transgene, leading to undesired
effects such as ectopic expression or even silencing (16, 17). To avoid these effects,
chromatin insulators can be used. These DNA elements, together with the proteins that bind
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to them, impair interactions with neighboring chromatin (18). Enhancers are used to
stimulate transcription and may do so in an orientation- and position-independent manner
(12). Locus control regions (LCRs) from globin or CD4 genes have been used as enhancers
for trangene expression in erythroid- or T cells, respectively (12, 19–21). For example, a
minimal human ε-globin promoter combined with a truncated human β-globin LCR, a
regulatory element that controls the erythroid-specific expression of all cis-linked globin
genes (22), have been used to generate mouse lines expressing GFP in the primitive
erythroid lineage (23, 24) (Figure 3A).

We recommend that expression of a newly designed transgene be tested in cultured cells

before moving forward with the generation of the transgenic mouse line. This precaution
helps to ensure that the transcriptional regulatory elements are functional and that the
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reporter sequence is translated to a functional protein.

1.2.2 Insertion of Exogenous DNA Into the Genome—The most commonly used
approaches for generation of fluorescent reporter mice are microinjection of DNA (plasmid
or BAC; see below) into a fertilized egg (Figure 2) and targeted insertion ("knock-in").
Insertion of a transgene into the mouse genome can also be achieved by viral infection of the
egg or injection of genetically modified embryonic stem (ES) cells into blastocysts (25).

In the “knock-in” method, DNA sequences are engineered to integrate directly into a defined
locus within the genome, using site-specific recombination (16). The knock-in approach
avoids problems related to random insertion, as expression of exogenous sequences is
controlled by the endogenous regulatory elements of the target gene. A recent example of a
knock-in transgenic mouse is the mirR-144/451-GFP line, in which GFP is expressed in
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adult and fetal liver erythroid cells (26, 27) (Figure 3). A concern for the knock-in approach
is that it may result in haploinsufficiency, influencing not only expression of the FP reporter
but also the phenotype of the resulting animal. For example, loss of one Runx1 allele
affected the distribution of HSCs in the embryo (28). This problem was overcome by linking
the sequences encoding the FP to the endogenous gene through an internal ribosomal entry
site (IRES) (29) to create a dicistronic fusion mRNA (30).

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Conventional transgenes are often too small to accurately reproduce the endogenous
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expression of the promoter/enhancer elements. Bacterial artificial chromosomes (BAC)

accommodate large DNA sequences, allowing cloning of all or most of the endogenous
regulatory elements required to recapitulate the normal pattern of gene expression when
linked to other sequences (31) such as those encoding a FP.

1.2.3 Alternative Approaches to Drive Hematopoietic Lineage-Specific

Expression of Fluorescent Protein Reporters—Inducible expression of fluorescent
reporters can be achieved using the CreloxP or FLP-FRT systems. For example, a mouse
line in which the fluorescent reporter sequences are preceded by a floxed STOP cassette
(e.g. refs. 32, 33, 34) can be mated with a deletor line designed to express Cre recombinase
under the control of a promotor active in the cells of interest. Such targeted recombination
approaches have been of great utility for lineage tracing studies (for a recent example, see
ref. 35).
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Depending on the system chosen, recombinase activity may be constitutive or inducible.

Inducible recombination activity can be triggered by tamoxifen in the case of a Creestrogen
receptor (ER) fusion gene or by doxycycline when Cre expresssion is controlled by a
Tetracycline (Tet) responsive element (reviewed in ref. 36). The Mx1-Cre deletor line can
be used for targeting of definitive hematopoietic lineages. In this system, Cre expression is
activated by injection of interferon or synthetic double-stranded RNA polyinosinic-
polycytidylic acid (poly I:C) (37).

1.3 Fluorescent Protein Reporters

A wide range of FPs covering nearly the entire visible spectrum can be used for generating
transgenic reporter mice (reviewed in refs. 2, 3, 4). Since the discovery of wtGFP (see
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Introduction), investigators have sought to generate other FPs FPs with reduced
phototoxicity, improved brightness and phosphostability over broad ranges of pH and
temperature (2–4). Site-directed mutagenesis of wtGFP has been employed not only for the
generation of FPs with improved functionality but also with diverse spectral characteristics,
for example cyan FP (CFP), blue FP (BFP), and yellow FP (YFP) (2–4).

FPs with emission peaks in the red and far-red spectra have been especially useful for to
live-cell or whole-animal imaging, owing to their long wavelength emission and,
consequently, reduced phototoxicity (2, 38). Initially, the tetrameric DsRed was cloned from
the nonbioluminescent sea anemone Discosoma striata but was found to be toxic to cells
(39). Subsequently, the monomeric variant mRFP was engineered and could be expressed
ubiquitously in mice without deleterious effects on development (40).
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Directed mutagenesis of mRFP was used to create several variants, including the orange
mTomato and the red mStrawberry and mCherry FPs (38). Far-red fluorophores such as
mPlum, genetically engineered from a blue chromoprotein of the sea anemone Actinia
equina (41), offer deep tissue penetration and reduced autofluorescence. Spectral properties
of various FPs are discussed in detail in refs. (2–4).

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1.3.1 Fluorescent Fusion Proteins—FPs can be engineered for localization to specific

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subcellular regions (42). For example, to localize the FP to the nucleus, a nuclear
localization signal is incorporated into the construct or the FP is fused to histone H2B
sequences. Histone H2B fusion-FPs bind to chromatin and are present through all phases of
the cell cycle (43, 44). Unlike cytoplasmic GFP, which is diluted during subsequent cell
divisions, H2B-FPs are stably expressed and permit monitoring of both the cell cycle and
apoptosis (44). A histone H2B-GFP expressed under the control of a human epsilon-globin
promoter and truncated LCR has been used for labeling the nuclei of primitive erythroid
cells (24) (Figure 3A). Cell morphology and migration can also be observed by labeling the
outer or inner leaflet of the cell membrane with lipid-tagged FP fusions (e.g. containing a
glycosylphospatidylinositol (GPI) anchor or a myristoylation sequence, ref. 45).

1.3.2 Photomodulatable FPs—Photoactivatable fluorescent proteins (PAFP) are

stimulated by lights of specific wavelengths, intensities and durations, allowing for
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spatiotemporal labeling of live cells, organelles and molecules (2–4). There are two types of
PAFP functions: photoactivation (PA) and photoswitching. Photoactivators convert from a
non-fluorescent to a bright fluorescent state and can be either irreversible or reversible. PA-
GFP is a GFP variant that is irreversibly converted to an anionic form, resulting in a 100-
fold increase in its emission intensity (46). Tetrameric kindling FP (KFP) can be reversibly
or irreversibly photoactivated, depending on the intensity of the activating light (4, 47). It
converts to a red fluorescent state following exposure to green light and returns to a non-
fluorescent state in the absence of stimulation (47). Photoswitchers change their fluorescent
state and emit at a different wavelength (such as cyan-green for PS-CFP or green-red for
EosFP, Kaede and Kikume Green-Red, KikGR) upon exposure to transient but intense light
(3).
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1.3.3 General Consideration for Choosing Fluorescent Protein Reporters—

When choosing an FP reporter protein, the investigator should consider whether it will be
used for multi-color analysis in combination with other FPs or in immunofluorescence
studies with a fluorophore of a different color. The availability of fluorescent variants allows
the researcher to select combinations that minimize spectral overlap. For example, the
combinations of GFP/CFP and GFP/YFP exhibit significant emission overlap, whereas
CFP/YFP does not (48). Bright reporters in the red or far-red spectra increase the
possibilities of combining different reporters to mark cells of different lineages or to mark
different regions of the same cell. Due to the lower phototoxicity of the excitation light
required by red or far-red fluorophores, these reporters are more suitable for live imaging
studies (49). While imaging or flow cytometric analysis of double transgenic mice
expressing FPs with overlapping spectra can be challenging (48), the judicious choice of
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excitation light and filters will allow optimal separation of reporter signals, as observed for
the simultaneous imaging of erythroid cells expressing ECFP and myeloid cells expressing
EGFP (50).

1.4 Confirmation and Analysis of Transgene Expression

1.4.1 Mouse background—The choice of genetic background should be carefully
considered in planning the generation of a transgenic mouse line. For microinjection,

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zygotes of mixed or outbred background are often used (16). Microinjection of zygotes from
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inbred mice is more difficult and embryo viability is lower (16).

Only a relatively small number among the many available inbred strains (e.g. C57BL/6 or
129/Sv) are routinely used to create transgenic or knockin mice. In the context of the present
discussion, inbred strains would be desirable if the transgenic animals will be mated with
knockout mice known to have a background dependent phenotype or if tissue from the
genetically modified animals will be used for HSC or other transplantation studies (51).
Genetic background effects (variable penetrance or expressivity) are caused by modifier
genes (51). ICR (CD1) mice are the most widely used outbred mouse strain. Unlike inbred
mice, ICR mice display inter-individual genetic variation. However, ICR mice are
inexpensive, have excellent reproductive and maternal characteristics, and yield relatively
large litter sizes (16).
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1.4.2 Breeding—Once founders are identified (see section 3.2), the colony should be
expanded. Female founders should be bred so that they can give birth to at least one litter
before being sacrificed. Male founders should be placed in a cage with two nontransgenic
females and plugs checked daily. Ideally, the male founder should plug 6–8 females in the
first few weeks (16). The gold standard for assessing transgene integration is germline
transmission to the F1 generation. By this metric, founders should transmit the transgene to
50% of their progeny. If transmission is not observed, the founder genotype should be
reanalyzed. If the founder is positive for the transgene and germline transmission does not
occur, it is likely that the founder is mosaic for the transgene and, therefore, either transmits
the transgene through the germline at very low levels or not at all. In certain scenarios,
transgenes will integrate at multiple loci resulting in progeny that inherit the transgene at
unusually high frequencies (52).
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In contrast with mouse lines created by gene targeting, each transgenic founder is distinct
because of the random nature of transgene integration. Therefore the decision to eliminate a
transgenic mouse line from a colony will be irreversible. To reduce costs, the investigator
may choose to maintain an active colony of a few mating pairs or a small number of males
that can be mated periodically to produce a younger generation. This is a relatively
inexpensive approach but carries the risk that transgene expression may decrease in later
generations or as the animals age; this phenomenon has often been seen for globin
transgenes (53). Transgene silencing may be avoided through preserving the line as frozen
embryos or sperm (so that in vitro fertilization can be performed at a later date) (51).
Cryopreservation services are provided by some institutional core transgenic mouse
facilities and by mouse suppliers such as Taconic Farms and Charles River Laboratory.
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1.5 Analysis of Fluorescent Protein Expression using Microscopy

The fluorescence of embryos, tissues or cells from transgenic reporter mice can be analyzed
using epifluorescence or confocal laser scanning microscopy. Confocal microscopes offer
several advantages over epifluorescence microscopes. Whereas in epifluorescent
microscopy, the entire field is illuminated by light emitted by a mercury or xenon UV lamp,
in confocal microscopy, light emitted by the laser is focused through a pinhole, creating

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point illumination. Out-of-focus signals are thereby eliminated and resolution is increased
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(54). The confocal microscope images thin sections of the specimen that can be combined
using the microscope’s software into accurate 3D reconstructions of the sample. Confocal
microscopes also have an increased level of sensitivity due to light detectors that can
amplify the signals received from the specimen. Another advantage of confocal microscopy
is that it is less invasive, resulting in reduced photobleaching (54). The illumination
provided by high-power lasers, combined with their reduced light scattering properties,
allows imaging of thick, semitransparent sections, live tissues or embryos (54). Newer
model epifluorescent microscopes use LED light sources that are more suitable for live
imaging than are classical mercury lamps.

For live imaging of explanted embryos or tissues, temperature and gas composition must be
carefully controlled using an environmental chamber. Inverted microscopes are typically
used to image live material. For a discussion of imaging mouse embryos using confocal
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microscopy, see refs. (55–57).

Regardless of the type of microscope used, it is essential to select the appropriate light
source and filters for analysis of the fluorescent specimen (54). The identity of the cells
expressing the FP may be determined using immunofluorescence, by staining for cell type-
specific markers. The staining can be performed on live or fixed cells in solution or on fixed
cells deposited on microscope slides. The fixation and permeabilization method should be
carefully optimized for each cell type. An overview of different fixation and
permeabilization options is reviewed in ref. (54).

1.6 Analysis of Fluorescent Protein Expression using Flow Cytometry

Analytical flow cytometry is a fundamental technique for assessing fluorescence in cells
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from transgenic FP reporter mice. Once tissues have been dispersed into single cell
suspensions, fluorescent reporter expression from a transgene, combined with antibody
staining for specific cell surface markers, can be analyzed using a flow cytometer to identify
the cell surface characteristics of the component cell populations. Fluorescence Activated
Cell Sorting (FACS) is a specialized type of flow cytometry that permits the physical
separation of a heterogeneous sample into distinct cell populations based on their
fluorescence. The ability to isolate labeled cell populations in a single step provides a
valuable tool for elucidation of their developmental potentials, cell cycle and other
properties, and for culture ex vivo. For detailed protocols and reviews, see ref. (58).

2. Materials
DNA purification for microinjection
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1. Agarose (Invitrogen; Cat # 16500).

2. Ethidium bromide (Sigma Aldrich; Cat # E7637).

3. Injection buffer: 10 mM Tris, pH 7.4, 0.2 mM EDTA.

4. QIAquick Gel Extraction Kit (Qiagen; Cat # 28704).

5. Restriction enzymes.

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Isolation of genomic DNA

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1. DirectPCR Lysis Reagent (mouse tail) (Viagen; Cat # 102-T).

2. Proteinase K (Invitrogen; Cat # 25530). Stock solution prepared at 10 mg/ml in

TrisHCl 20 mM, pH 8. Aliquots are stored at −20°C.

Polymerase chain reaction (PCR)

1. Plastic tubes for PCR, 0.2 ml (Denville Scientific, Cat. # C18063).

2. TaKaRa ExTaq DNA polymerase, supplied with 10X ExTaq buffer and dNTP mix
(Clontech, e.g. Cat. # RR001A).

3. Thermal cycler (available from a variety of companies).

4. For agarose gel electrophoresis: agarose, gel casting tray, comb with desired
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number of teeth, power supply, ethidium bromide, DNA size ladder.

Dissecting tools
1. Dissecting scissors (Roboz Surgical Instrument Inc., Gaithersburg, MD; Cat #
RS-6702 and RS-5882) and forceps (Sigma Aldrich, St. Louis, MO; Cat # F4267).

2. Watchmaker’s forceps, Dumont #5 and #55 (Roboz, FST).

3. Sterile plastic transfer pipettes, 3 ml (VWR; Cat # 414004-037).

4. Stereomicroscope with transmitted and reflected light sources (Zeiss, Leica or

Nikon).

Glassware and Plasticware

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1. Nunclon tissue culture plates, 24 well (Nunc, Thermo Fisher Scientific; Cat #
142475).

2. Circular coverslips, 12 mm, no. 1 (Thermo Fisher Scientific; Cat # 12-545-80).

3. 3 and 5 ml syringes (BD Biosciences; Cat # 309657 and # 309646).

4. Syringe needles, 20G and 25G (BD Biosciences; Cat # 305176 and # 305122).

5. Polypropylene tubes, 15 and 50 ml (Corning, Lowell, MA; Cat # 430766 and #

430291).

Embryo dissection and cell preparation for flow cytometry

1. Phosphate buffered saline (PBS) pH 7.4 (GIBCO Invitrogen, Carlsbad, CA; Cat #
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10010-023).

2. Iscove’s Modified Dulbecco’s Medium (GIBCO Invitrogen; Cat # 12440-079).

3. Fetal Bovine Serum (FBS; Hyclone, Thermo Fisher Scientific).

4. Dissection medium: IMDM +10% FBS.

5. Heparin (Sigma Aldrich, St. Louis, MI; Cat # H3149): Dissolve in PBS to 12.5
mg/ml to produce a stock solution (100X).

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6. BD Falcon 40 μm and 70 μm cell strainers (BD Biosciences, San Jose, CA; Cat #
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352340 and # 352350).

7. Cell Dissociation Buffer (GIBCO Invitrogen; Cat # 13150-016).

8. Collagenase (Sigma Aldrich; Cat # C2674): Stock solution prepared at 100 mg/ml
in medium supplemented with 20% serum. Aliquots are stored at −20°C.

Flow cytometry
1. FACS buffer: heat-inactivated FBS diluted in PBS (see Note 1).

2. DAPI (4',6-diamidino-2-phenylindole dihydrochloride; Sigma Aldrich; Cat #

D9542);

3. Propidium iodide (Sigma-Aldrich; Cat. # P4170). Dilute powder in distilled water

to prepare 1000X stock solution.
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Immunostaining and microscopy

1. 4% Paraformaldehyde (PFA) obtained by dilution of 16% PFA (Electron
Microscopy Sciences, Cat # 15710) in PBS.

2. Washing buffers: PBS with 0.05% Tween-20 (v/v) (Sigma Aldrich; Cat # P1379)
(PBST); PBST with 0.05% no-fat skim milk powder (Carnation)(PBSMT).

3. Vectashield with DAPI (Vector Labs, Burlingame, CA; Cat # H-1500) or without
(Vector Labs, Burlingame, CA; Cat # H-1400).

4. Primary and secondary antibodies of choice.

5. Triton X-100 (Sigma Aldrich; Cat # T8787).

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6. Bovine serum albumin (BSA) (Sigma Aldrich; Cat # B4287).

7. Blocking buffer: 2% BSA, 0.1% Triton X-100 in PBS.

3. Methods
3.1 DNA Preparation for Microinjection
Transgenes are designed so that the gene to be microinjected can be excised and purified
away from plasmid sequences. Prokaryotic sequences from the plasmid do not appear to
influence the efficiency of transgene integration but they may impair expression from
eukaryotic sequences (14, 15, 16). Therefore, the plasmid backbone sequences should be
excised from the transgene construct. It is important to include a final purification step to
remove particulate material that may clog the injection needle. The following protocol yields
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clean DNA for microinjection from plasmids smaller than 20 kb. Plasmids larger than 20 kb
require a different purification procedure (59), that will not be discussed here.

1FACS buffer is PBS containing proteins (e.g. FBS). The proteins in FBS reduce cell loss by decreasing their interaction with plastic
during the antibody staining procedure and, more importantly, the plastic tubing of the fluidics system of the flow cytometer. During
antibody staining, we routinely use PBS containing 10% heat-inactivated FBS (10% FACS buffer). After the last washing step, the
cells are resuspended in PBS containing 3% heat-inactivated FBS (3% FACS buffer).

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1. Digest plasmid DNA to completion with the appropriate restriction enzyme(s).

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2. Separate the restriction fragments by electrophoresis through an agarose gel (see

Note 2).

3. Using an ultraviolet transilluminator, identify and isolate the band containing the
transgene to be microinjected.

4. Extract and purify the DNA according to the manufacturer’s protocols for the
QIAquick Gel Extraction Kit (QIAGEN).

5. Elute the DNA from the QIAquick column using injection buffer (see Materials
section) and measure the DNA concentration using a NanoDrop spectrophotometer
(Thermo Scientific).

6. Pronuclear injection is typically performed in an institutional core transgenic

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mouse facility. For a detailed protocol, see ref. (60).

At our institution, the purified DNA (1–2 μg, 50–100 ng/μl) is submitted to the Mouse
Genetics and Gene Targeting Shared Resource Facility. Filtration and final dilution of the
DNA to be used for microinjection is performed by the facility, as is transfer of the injected
zygote into pseudopregnant females(16).

3.2 Genotyping
Offspring born from injected zygotes are termed "founders" and are usually screened for the
presence of the transgene. Genomic screening is most commonly performed using
polymerase chain reaction (PCR) analysis of DNA from biopsied tissue (for comments on
primer design, see Note 3). In most cases, the microinjected DNA will be stably integrated
at the one-cell stage. However, the foreign DNA may integrate at a later (e.g. 4- or 8-cell)
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stage, resulting in mosaic expression of the transgene and disruption of germline

transmission (16). In addition, silencing of the transgene may occur following integration
into or near heterochromatin (see Note 4).

It may be possible to identify founders by microscopic analysis of biopsied tissues (see Note
5).

Preparation of Tissue Samples for Genotyping

1. Tail tips <0.5 cm may be biopsied from pups ≤ 21 days old without the use of an
analgesic. Adult mice must be anesthetized according to federal and Institutional

2Some investigators prefer to stain the DNA with crystal violet to avoid toxicity of ethidium bromide.
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3PCR sequences for common fluorescent reporters such as GFP are available in the literature (refer to Table 2 for primer sequences
used in our laboratory). If more than one FP transgenic reporter mouse line is maintained in the colony, it is advisable to use a primer
specific for sequences outside the FP coding region. For example, one primer would hybridize with a sequence in the FP coding
region while the second would hybridize with an external sequence such as the promoter or 3'-UTR.
4Microinjected DNA can be transcriptionally silenced as a result of integration in or near heterochromatin, a process called
variegation. Variegation is a heterocellular pattern of gene expression sometimes observed in transgenic mice. It is commonly age-
dependent and also results from progressive breeding of the mice (53). This phenomenon is often seen with both the alpha- and beta-
globin genes. Variegation may be suppressed if the transgene is linked to certain globin gene enhancer elements (67, 68).
5Identification of founders may be possible using direct microscopic analysis of tissue biopsies from pups (up to 21 days old) if
transgene expression is sufficiently bright. For example, we genotype Flk1-H2B-YFP transgenic mice (69) by examining ear clips
from 10 day-old newborn mice. The Flk1 promoter is active in endothelial cells of the skin at this stage. This simple genotyping
approach can be applied to other transgenic fluorescent reporters that are expressed in tissues that are easily collected by biopsy.

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Animal Care and Use Committee (IACUC) regulations. Using scissors cleaned
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with 70% ethanol, cut a 1cm section of the tail tip of the founder mouse. Be sure to
wipe the scissors with 70% ethanol before cutting the next tail, to prevent DNA
cross-contamination between samples. Place the tail tip samples into labeled 1.5 ml
eppendorf tubes. Samples can be stored frozen at −80°C and DNA isolated at a
later date.

2. For DNA isolation, add 195 μl of DirectPCR Lysis Reagent (mouse tail) to each
tube, followed by 5 μl Proteinase K (10 mg/ml).

3. Incubate the tubes at 55°C using mild agitation for 4 hr or overnight.

4. Heat inactivate the samples at 85°C for 45 min.

Genomic PCR
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1. For PCR, pipette 1 μl DNA into a clean 0.2 ml tube containing 0.5 μl of 10 μM
stocks of forward and reverse primer (see Table 2); 2 μl of 10X ExTaq Buffer
containing MgCl2 (supplied with the polymerase), 1 μl of 2.5 mM dNTP mix, 0.2
μl TaKaRa ExTaq DNA polymerase (Clontech), and water to a total volume of 20
μl. For multiple reactions using the same forward and reverse primers, it is
advisable to prepare a master mix containing all components except for the
genomic DNA. See Table 2 for fluorescent reporter primer sequences used in our
laboratory.

2. Conditions for PCR in a standard thermal cycler: (a) initial denaturation at 95°C for
5 min; (b) 35 cycles of: denaturation at 95°C for 30 sec, annealing at 55°C for 30
sec, and extension at 72°C for 1 min; (c) final extension at 72°C for 5 min. Samples
are then maintained at 4°C until ready for analysis using standard agarose gel
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electrophoresis (e.g. ref. 61). Annealing temperature is generally close to the

melting temperature for the primers and may need to be optimized. The extension
time may also need to be optimized according to the length of the PCR product.

3.3 Dissection
Following identification of founders using genomic PCR, expression of the reporter is
examined in whole embryos, dissected tissues, peripheral blood or bone marrow using
microscopy or flow cytometry.

3.3.1 Embryo dissection—Typically the analysis of hematopoietic lineages in the

embryo/fetus is performed from E7.5 through E16.5. Below we present a general protocol
for dissection of E9.5 to E14.5 embryos. For more detailed information about dissection of
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earlier stage embryos, see refs. (16, 62).

1. Euthanize pregnant mother by CO2 asphyxiation followed by cervical dislocation.

2. Spray the abdomen of the mouse with 70% ethanol. Pinch the fur on the abdomen
and make a midline incision to open the abdominal cavity and expose the uterine
horns.

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 Vacaru et al. Page 12

3. Use a forceps to lift up the ends of the uterine horns and carefully remove the
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attached connective tissue and fat. Wash off the maternal blood in a 10 cm Petri
dish containing PBS supplemented with 5% FBS.

4. Carefully cut each conceptus and transfer it to a 10 cm Petri dish containing PBS.
Rinse thoroughly with PBS to remove maternal blood and then transfer to a Petri
dish containing dissection medium.

5. Using a pair of #5 or #55 watchmaker’s forceps, peel away the uterine tissue,
starting from the incision created in step 4.

6. Gently detach the Reichert’s membrane by holding the embryo with one pair of
forceps and removing the membrane with a second pair of forceps.

7. The embryo is visible inside the yolk sac, with the placenta attached. The placenta
can be separated using forceps or fine scissors. Once the placental vessels are cut,
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the embryonic blood will be released into the medium. Placental dissociation is
performed as described in section 3.3.4. The YS can be removed from the embryo
by carefully peeling it away using forceps.

3.3.2 Isolation of Peripheral Blood from Embryos—Peripheral blood can be

obtained from ~E9.5 onward (63), when the embryonic circulation is well established (64).

1. Each embryo (YS and placenta intact) is transferred to a well of a 24-well dish
containing dissection medium with 0.5% heparin. Fluorescence can be easily
evaluated using a fluorescence stereomicroscope.

2. After selection of the desired embryos, peripheral blood can be collected. Grab the
region between the embryo and the placenta using a #5 watchmaker’s forceps and
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cut umbilical and vitelline vessels with another #5 forceps. Peripheral blood cells
will be released into the medium in large numbers.

3. Allow the embryos to exsanguinate (~10 min). Blood cells will collect at the
bottom of the well.

4. Remove the embryos and debris from each well and resuspend the blood cells in
dissection medium, using a P1000 Pipetman.

5. Filter the blood cell suspension through a 40 μm cell strainer and then collect the
cells by centrifugation at 1200 rpm (100 × g) in an Eppendorf microcentrifuge.

6. Resuspend the cells at the desired concentration in a buffer appropriate for the
intended application.
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3.3.3 Dissection of Fetal Liver—The FL does not develop a tightly adherent epithelial
structure and can be easily dispersed mechanically, allowing simple isolation of
hematopoietic cells through ~E16.5.

1. Carefully dissect the FL from the embryo using a pair of #5 watchmaker’s forceps.

2. Transfer the FL to a 1.5 ml eppendorf tube containing 0.5 ml dissection medium.

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 Vacaru et al. Page 13

3. Disperse the FL by pipetting in dissection medium using a P1000 Pipetman until

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obtaining a homogeneous suspension.

4. Filter the cell suspension through a 70μm cell strainer, collect the cells by
centrifugation at 1200 rpm (100 × g) in an Eppendorf microcentrifuge and
resuspend in PBS for cytocentrifugation or in FACS buffer for flow cytometry.

3.3.4 Dissection of Yolk Sac and Placenta—YS and placenta contain endothelial cells
with adherent junctions and endodermal cells with tight junctions, therefore requiring more
vigorous dissociation steps than for FL prior to isolation of hematopoietic cells.

1. Dissect the YS (62) and placenta (65) from each embryo at the desired stage.

2. Place each tissue into an individual 1.5 ml eppendorf tube containing 1 ml

collagenase. If two or more YSs or placentae will be collected from embryos at the
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same stage, they can be pooled in a 15 ml conical tube containing 4 ml collagenase.

Dissociation to single cells is more efficient if narrow dissection scissors (Roboz
RS-6702) are used to macerate the YS or placental tissue.

3. Incubate at 37°C for at least 20 min. Shake vigorously every 5 min. until a uniform
cell suspension has been obtained (no large clumps remain). Filter the sample
through a 70μm cell strainer, collect by centrifugation in an Eppendorf
microcentrifuge at 1200 rpm (100 × g) and resuspend in PBS or FACS buffer, as
described above.

3.3.5 Isolation of the AGM Region—Dissection of the AGM region has been described
in detail, and presented in a video, by others (66).
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3.3.6 Isolation of Adult Bone Marrow

1. Euthanize the adult mouse by CO2 asphyxiation followed by cervical dislocation.

2. Spray the abdomen of the mouse with 70% ethanol. Make a midline incision to
open the abdominal cavity and expose the hind legs.

3. Remove all the muscle and connective tissue from the bones using scissors and
then cut the tibia and femur from the joints.

4. Cut the ends of the bones and flush the bone marrow with 1 ml buffer of choice
(according to the subsequent analysis) into a collection tube stored on ice, using a
25G needle and a 5 ml syringe.

5. Homogenize the bone marrow suspension by pipetting up and down and pass the
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cell suspension through a 70 μm cell strainer.

3.4 General Immunofluorescence Protocol

1. Fix and permeabilize the cells using the appropriate reagents (e.g. 4% PFA in PBS;
see Note 6). Wash the cells twice for 5 min in PBS.

2. Block the cells for 15–30 min in blocking buffer.

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 Vacaru et al. Page 14

3. Dilute primary antibody in blocking buffer and add 100 μl per slide. Incubate for 1
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hr at room temperature.

4. Wash 3 times for 5 min with PBS + 0.01 % (v/v) Triton X-100.

5. Dilute secondary antibody in blocking buffer and add 100 μl per slide. Incubate for
45 min.

6. Wash 3 times for 5 min with PBS + 0.01 % (v/v) Triton X-100. Rinse in PBS and
then in water to avoid crystallization of the salts from PBS.

7. Mount coverslips using Vectashield mounting medium with or without DAPI (see
Note 7).

3.5 Flow Cytometry

3.5.1 Labeling of Cells for Flow Cytometry
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1. Count cells using a hemacytometer and dispense 1×106 cells into a 1.5 ml
eppendorf tube. Collect by centrifugation at 1200 rpm (100 × g) in an Eppendorf
microcentrifuge and aspirate supernatant.

2. Dilute fluorescently conjugated antibody into 10% FACS buffer (see Note 1). We
generally use 2 μg of antibody per 100,000 cells; however, each antibody should be
titrated for optimal results. Antibodies conjugated to different fluorochromes
should be combined in the same "cocktail" to minimize cell loss due to additional
incubation and washing steps.

3. As EryP do not express Fc receptors, they exhibit low background binding to

antibodies. We have not found that treatment of cells with normal mouse serum or
FcBlock is necessary. Cells expressing the Fc receptor should be treated with
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normal mouse serum or purified anti-mouse CD16/CD32 to prevent non-specific

binding.

4. Resuspend cells in a 1.5 ml Eppendorf tube containing 100 μl of the antibody

cocktail and incubate on ice in the dark for 20 min, inverting the tubes every 5 min.

5. Wash with 1 ml 10% FACS buffer and collect cells by centrifugation at 1200 rpm
(100 × g) in an Eppendorf microcentrifuge.

6. If primary antibodies are unconjugated or biotin-conjugated, treat cells with

fluorescently conjugated secondary antibodies or streptavidin antibodies in a final
volume of 100 μl in 10% FACS buffer. Resuspend the cell pellet in 100 μl of
diluted streptavidin. Incubate on ice in the dark for 20 min., inverting the tube
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every 5 min.

6Exposure of the FPs to fixation reagents (e.g. formaldehyde, glutaraldehyde, methanol, acetone) denatures protein, leading to loss of
fluorescence. Therefore, the fixation method (reagent and time of exposure) must be optimized. When using PFA or glutaraldehyde as
fixative, quenching for 15 min with 0.1M glycine (final concentration) following fixation will help to reduce FP denaturation. If
fluorescence is lost during the fixation step, the FP can be detected by immunofluorescece after staining with an FP specific antibody.
7The use of Vectashield mounting medium with DAPI may result in increased background fluorescence. This may present a particular
problem when the intensity of reporter fluorescence is weak. It may, therefore, be desirable to stain with DAPI, followed by washing
of the slide and mounting the coverslip using Vectashield without DAPI.

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 Vacaru et al. Page 15

7. Wash with 1 ml 10% FACS buffer and collect cells by centrifugation at 1200 rpm
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(100 × g) in an Eppendorf microcentrifuge..

8. Resuspend the cell pellet in 400 μl of 3% FACS buffer containing DAPI (see Note
8) if a UV laser available or PI if a UV laser is not available. Transfer the cell
suspension to a 5 ml round bottom tube (see Note 9).

9. Analyze using a flow cytometer.

3.5.2 Preparation of Cells for Sorting by FACS

1. Label cells for FACS as described above in section 3.5.1.

2. Prepare collection tubes for sorted cells. If the sorted cells are to be cultured,
collect into a 5 ml round bottom tube containing 1 ml sterile medium.

3. FACS instruments are typically operated by trained personnel. The investigator will
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advise the operator regarding the populations to be sorted.

4. Tubes containing sorted cells are kept briefly on ice until the cells can be collected
by centrifugation. Cell pellets can be stored frozen at -80ºC prior to isolation of
RNA.

Acknowledgments
We thank J. Barminko and G. Camprecios for their comments on the manuscript. Work in our laboratory was
supported in part by grants to M.H.B. from the National Institutes of Health (RO1 HL62248 and, DK52191, and
EB02209), the Roche Foundation for Anemia Research (grant 9699367999, cycle X) and the New York State
Department of Health (NYSTEM grant N08G-024).

References
Author Manuscript

1. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ. Primary structure of the
Aequorea victoria green-fluorescent protein. Gene. 1992 Feb 15; 111(2):229–33. [PubMed:
1347277]
2. Shaner NC, Steinbach PA, Tsien RY. A guide to choosing fluorescent proteins. Nat Methods. 2005
Dec; 2(12):905–9. [PubMed: 16299475]
3. Nowotschin S, Eakin GS, Hadjantonakis AK. Live-imaging fluorescent proteins in mouse embryos:
multi-dimensional, multi-spectral perspectives. Trends Biotechnol. 2009 May; 27(5):266–76.
[PubMed: 19339068]
4. Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA. Fluorescent proteins and their applications
in imaging living cells and tissues. Physiol Rev. 2010 Jul; 90(3):1103–63. [PubMed: 20664080]
5. Orkin SH, Zon LI. Hematopoiesis: an evolving paradigm for stem cell biology. Cell. 2008 Feb 22;
132(4):631–44. [PubMed: 18295580]
6. Baron MH, Isern J, Fraser ST. The embryonic origins of erythropoiesis in mammals. Blood. 2012
Author Manuscript

May 24; 119(21):4828–37. [PubMed: 22337720]

8To exclude dead cells, which may bind nonspecifically to antibodies and produce a false positive signal, we routinely resuspend the
final cell pellet in FACS buffer containing 0.2 mg/ml DAPI. DAPI is a poorly cell-permeable DNA binding dye that is excited by the
violet laser in the flow cytometer. DAPI is soluble in water but not in PBS. Therefore, we prepare a 1000X stock solution in deionized
water. The stock is then diluted into FACS buffer.
9Sorting time and efficiency varies widely due to concentration of cell suspension and the degree of cell death. A highly concentrated
sample of cells and significant amount of cell death will increase the "abortion rate" – the rate at which droplets containing single cells
are not selected by the FACS machine to be sorted –resulting in lower recovery of viable sorted cells. The abortion rate can be reduced
and sample purity increased by diluting the sample and/or by modifying the cell dissociation technique.

Methods Mol Biol. Author manuscript; available in PMC 2015 May 04.
 Vacaru et al. Page 16

7. Palis J, Yoder MC. Yolk-sac hematopoiesis: the first blood cells of mouse and man. Exp Hematol.
2001 Aug; 29(8):927–36. [PubMed: 11495698]
Author Manuscript

8. Dzierzak E, Speck NA. Of lineage and legacy: the development of mammalian hematopoietic stem
cells. Nat Immunol. 2008 Feb; 9(2):129–36. [PubMed: 18204427]
9. Orkin SH, Zon LI. SnapShot: hematopoiesis. Cell. 2008 Feb 22.132(4):712. [PubMed: 18295585]
10. Kozak M. At least six nucleotides preceding the AUG initiator codon enhance translation in
mammalian cells. J Mol Biol. 1987 Aug 20; 196(4):947–50. [PubMed: 3681984]
11. Zufferey R, Donello JE, Trono D, Hope TJ. Woodchuck hepatitis virus posttranscriptional
regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol. 1999
Apr; 73(4):2886–92. [PubMed: 10074136]
12. Bulger M, Groudine M. Enhancers: the abundance and function of regulatory sequences beyond
promoters. Dev Biol. 2010 Mar 15; 339(2):250–7. [PubMed: 20025863]
13. Choi T, Huang M, Gorman C, Jaenisch R. A generic intron increases gene expression in transgenic
mice. Mol Cell Biol. 1991 Jun; 11(6):3070–4. [PubMed: 2038318]
14. Chada K, Magram J, Raphael K, Radice G, Lacy E, Costantini F. Specific expression of a foreign
β-globin gene in erythroid cells of transgenic mice. Nature. 1985; 314:377–80. [PubMed:
Author Manuscript

3982505]
15. Townes TM, Lingrel JB, Chen HY, Brinster RL, Palmiter RD. Erythroid-specific expression of
human beta-globin genes in transgenic mice. EMBO J. 1985 Jul; 4(7):1715–23. [PubMed:
2992937]
16. Nagy, A.; Gertsenstein, M.; Vintersten, K.; Behringer, R. Manipulating the Mouse Embryo: A
Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2003.
17. Henikoff S. Conspiracy of silence among repeated transgenes. Bioessays. 1998 Jul; 20(7):532–5.
[PubMed: 9723001]
18. Bushey AM, Dorman ER, Corces VG. Chromatin insulators: regulatory mechanisms and
epigenetic inheritance. Mol Cell. 2008 Oct 10; 32(1):1–9. [PubMed: 18851828]
19. Festenstein R, Tolaini M, Corbella P, Mamalaki C, Parrington J, Fox M, et al. Locus control region
function and heterochromatin-induced position effect variegation. Science. 1996 Feb 23;
271(5252):1123–5. [PubMed: 8599090]
20. Li Q, Harju S, Peterson KR. Locus control regions: coming of age at a decade plus. Trends Genet.
Author Manuscript

1999 Oct; 15(10):403–8. [PubMed: 10498936]

21. Festenstein R, Kioussis D. Locus control regions and epigenetic chromatin modifiers. Curr Opin
Genet Dev. 2000 Apr; 10(2):199–203. [PubMed: 10753778]
22. Forrester WC, Novak U, Gelinas R, Groudine M. Molecular analysis of the human beta-globin
locus activation region. Proc Natl Acad Sci U S A. 1989 Jul; 86(14):5439–43. [PubMed: 2748594]
23. Dyer MA, Farrington SM, Mohn D, Munday JR, Baron MH. Indian hedgehog activates
hematopoiesis and vasculogenesis and can respecify prospective neurectodermal cell fate in the
mouse embryo. Development. 2001 May; 128(10):1717–30. [PubMed: 11311154]
24. Isern J, Fraser ST, He Z, Baron MH. The fetal liver is a niche for maturation of primitive erythroid
cells. Proc Natl Acad Sci U S A. 2008 May 6; 105(18):6662–7. [PubMed: 18445648]
25. Haruyama N, Cho A, Kulkarni AB. Overview: engineering transgenic constructs and mice. Curr
Protoc Cell Biol. 2009 Mar.Chapter 19(Unit 19 0)
26. Rasmussen KD, O'Carroll D. The miR-144/451eGFP allele, a novel tool for resolving the erythroid
potential of hematopoietic precursors. Blood. 2011 Sep 15; 118(11):2988–92. [PubMed:
Author Manuscript

21791432]
27. Buza-Vidas N, Cismasiu VB, Moore S, Mead AJ, Woll PS, Lutteropp M, et al. Dicer is selectively
important for the earliest stages of erythroid development. Blood. 2012 Sep 20; 120(12):2412–6.
[PubMed: 22869792]
28. North TE, de Bruijn MF, Stacy T, Talebian L, Lind E, Robin C, et al. Runx1 expression marks
long-term repopulating hematopoietic stem cells in the midgestation mouse embryo. Immunity.
2002 May; 16(5):661–72. [PubMed: 12049718]

Methods Mol Biol. Author manuscript; available in PMC 2015 May 04.
 Vacaru et al. Page 17

29. Lorsbach RB, Moore J, Ang SO, Sun W, Lenny N, Downing JR. Role of RUNX1 in adult
hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage
Author Manuscript

expression. Blood. 2004 Apr 1; 103(7):2522–9. [PubMed: 14630789]

30. Mountford PS, Smith AG. Internal ribosome entry sites and dicistronic RNAs in mammalian
transgenesis. Trends Genet. 1995 May; 11(5):179–84. [PubMed: 7540337]
31. Vintersten K, Testa G, Naumann R, Anastassiadis K, Stewart AF. Bacterial artificial chromosome
transgenesis through pronuclear injection of fertilized mouse oocytes. Methods Mol Biol. 2008;
415:83–100. [PubMed: 18370149]
32. Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM, et al. Cre reporter strains
produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC developmental
biology. 2001; 1:4. [PubMed: 11299042]
33. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter
mouse. Genesis. 2007 Sep; 45(9):593–605. [PubMed: 17868096]
34. Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, et al. A robust and high-
throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci.
2010 Jan; 13(1):133–40. [PubMed: 20023653]
Author Manuscript

35. Boyer SW, Beaudin AE, Forsberg EC. Mapping differentiation pathways from hematopoietic stem
cells using Flk2/Flt3 lineage tracing. Cell Cycle. 2012 Sep 1; 11(17):3180–8. [PubMed:
22895180]
36. Lewandoski M. Analysis of Mouse Development with Conditional Mutagenesis. Handbook of
Experimental Therapeutics. 2007; 178:231–58.
37. Kuhn R, Schwenk F, Aguet M, Rajewsky K. Inducible gene targeting in mice. Science. 1995 Sep
8; 269(5229):1427–9. [PubMed: 7660125]
38. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. Improved
monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red
fluorescent protein. Nat Biotechnol. 2004 Dec; 22(12):1567–72. [PubMed: 15558047]
39. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, et al. A monomeric red
fluorescent protein. Proc Natl Acad Sci U S A. 2002 Jun 11; 99(12):7877–82. [PubMed:
12060735]
40. Zhu H, Wang G, Li G, Han M, Xu T, Zhuang Y, et al. Ubiquitous expression of mRFP1 in
Author Manuscript

transgenic mice. Genesis. 2005 Jun; 42(2):86–90. [PubMed: 15880439]

41. Shkrob MA, Yanushevich YG, Chudakov DM, Gurskaya NG, Labas YA, Poponov SY, et al. Far-
red fluorescent proteins evolved from a blue chromoprotein from Actinia equina. Biochem J. 2005
Dec 15; 392(Pt 3):649–54. [PubMed: 16164420]
42. Snapp E. Design and use of fluorescent fusion proteins in cell biology. Curr Protoc Cell Biol. 2005
Jul.Chapter 21(Unit 21 4)
43. Kanda T, Sullivan KF, Wahl GM. Histone-GFP fusion protein enables sensitive analysis of
chromosome dynamics in living mammalian cells. Curr Biol. 1998 Mar 26; 8(7):377–85.
[PubMed: 9545195]
44. Hadjantonakis AK, Papaioannou VE. Dynamic in vivo imaging and cell tracking using a histone
fluorescent protein fusion in mice. BMC Biotechnol. 2004 Dec 24.4:33. [PubMed: 15619330]
45. Rhee JM, Pirity MK, Lackan CS, Long JZ, Kondoh G, Takeda J, et al. In vivo imaging and
differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice.
Genesis. 2006 Apr; 44(4):202–18. [PubMed: 16604528]
46. Lukyanov KA, Chudakov DM, Lukyanov S, Verkhusha VV. Innovation: Photoactivatable
Author Manuscript

fluorescent proteins. Nat Rev Mol Cell Biol. 2005 Nov; 6(11):885–91. [PubMed: 16167053]
47. Chudakov DM, Belousov VV, Zaraisky AG, Novoselov VV, Staroverov DB, Zorov DB, et al.
Kindling fluorescent proteins for precise in vivo photolabeling. Nat Biotechnol. 2003 Feb; 21(2):
191–4. [PubMed: 12524551]
48. Stadtfeld M, Varas F, Graf T. Fluorescent protein-cell labeling and its application in time-lapse
analysis of hematopoietic differentiation. Methods Mol Med. 2005; 105:395–412. [PubMed:
15492410]

Methods Mol Biol. Author manuscript; available in PMC 2015 May 04.
 Vacaru et al. Page 18

49. Nowotschin S, Eakin GS, Hadjantonakis AK. Dual transgene strategy for live visualization of
chromatin and plasma membrane dynamics in murine embryonic stem cells and embryonic tissues.
Author Manuscript

Genesis. 2009 May; 47(5):330–6. [PubMed: 19358158]

50. Heck S, Ermakova O, Iwasaki H, Akashi K, Sun CW, Ryan TM, et al. Distinguishable live
erythroid and myeloid cells in beta-globin ECFP × lysozyme EGFP mice. Blood. 2003 Feb 1;
101(3):903–6. [PubMed: 12393544]
51. Papaioannou, VE.; Behringer, RR. Mouse Phenotypes: A Handbook of Mutation Analysis. Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2005.
52. Conner DA. Transgenic mouse colony management. Curr Protoc Mol Biol. 2005 Aug.Chapter
23(Unit 23):10. [PubMed: 18265361]
53. Robertson G, Garrick D, Wilson M, Martin DI, Whitelaw E. Age-dependent silencing of globin
transgenes in the mouse. Nucleic Acids Res. 1996 Apr 15; 24(8):1465–71. [PubMed: 8628679]
54. Spector, DL.; Goldman, RD. Basic Methods in Microscopy. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory Press; 2006.
55. Jones EA, Baron MH, Fraser SE, Dickinson ME. Dynamic in vivo imaging of mammalian
hematovascular development using whole embryo culture. Methods Mol Med. 2005; 105:381–94.
Author Manuscript

[PubMed: 15492409]
56. Udan, RS.; Dickinson, ME. Imaging Mouse Embryonic Development. In: Wassarman, PM.;
Soriano, PM., editors. Methods in Enzymology. San Diego, CA: Academic Press; 2010. p.
329-249.
57. Nowotschin, S.; Ferrer-Vaquer, A.; Hadjantonakis, A-K. Imaging Mouse Development with
Confocal Time-Lapse Microscopy. In: Wassarman, PM.; Soriano, PM., editors. Methods in
Enzymology. San Diego, CA: Academic Press; 2010. p. 351-77.
58. Hawley, TS.; Hawley, R. Flow Cytometry Protocols. 3. Totowa, NJ: Humana Press; 2011.
59. Krajnc NL, Smrekar F, Cerne J, Raspor P, Modic M, Krgovic D, et al. Purification of large
plasmids with methacrylate monolithic columns. J Sep Sci. 2009 Aug; 32(15–16):2682–90.
[PubMed: 19598166]
60. Ittner LM, Gotz J. Pronuclear injection for the production of transgenic mice. Nat Protoc. 2007;
2(5):1206–15. [PubMed: 17546016]
61. Sambrook, J.; Fritsch, EF.; Maniatis, T. Molecular Cloning: A Laboratory Manual. 2. Cold Spring
Author Manuscript

Harbor: Cold Spring Harbor Laboratory Press; 1989.

62. Baron MH, Mohn D. Mouse embryonic explant culture system for analysis of hematopoietic and
vascular development. Methods Mol Med. 2005; 105:231–56. [PubMed: 15492399]
63. Fraser ST, Isern J, Baron MH. Maturation and enucleation of primitive erythroblasts during mouse
embryogenesis is accompanied by changes in cell-surface antigen expression. Blood. 2007 Jan 1;
109(1):343–52. [PubMed: 16940424]
64. McGrath KE, Koniski AD, Malik J, Palis J. Circulation is established in a stepwise pattern in the
mammalian embryo. Blood. 2003 Mar 1; 101(5):1669–76. [PubMed: 12406884]
65. Gekas C, Rhodes KE, Mikkola HK. Isolation and visualization of mouse placental hematopoietic
stem cells. Current protocols in stem cell biology. 2008 Aug; Chapter 2(Unit 2A 8 1–2A):8–14.
[PubMed: 18729047]
66. Morgan K, Kharas M, Dzierzak E, Gilliland DG. Isolation of Early Hematopoietic Stem Cells from
Murine Yolk Sac and AGM. J Vis Exp. 2008; 16:e789.
67. Graubert TA, Hug BA, Wesselschmidt R, Hsieh CL, Ryan TM, Townes TM, et al. Stochastic,
stage-specific mechanisms account for the variegation of a human globin transgene. Nucleic Acids
Author Manuscript

Res. 1998 Jun 15; 26(12):2849–58. [PubMed: 9611227]

68. Robertson G, Garrick D, Wu W, Kearns M, Martin D, Whitelaw E. Position-dependent variegation
of globin transgene expression in mice. Proc Natl Acad Sci U S A. 1995 Jun 6; 92(12):5371–5.
[PubMed: 7777514]
69. Fraser ST, Hadjantonakis AK, Sahr KE, Willey S, Kelly OG, Jones EA, et al. Using a histone
yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice.
Genesis. 2005 Jul; 42(3):162–71. [PubMed: 15986455]

Methods Mol Biol. Author manuscript; available in PMC 2015 May 04.
 Vacaru et al. Page 19

70. Suzuki N, Ohneda O, Minegishi N, Nishikawa M, Ohta T, Takahashi S, et al. Combinatorial Gata2
and Sca1 expression defines hematopoietic stem cells in the bone marrow niche. Proc Natl Acad
Author Manuscript

Sci U S A. 2006 Feb 14; 103(7):2202–7. [PubMed: 16461905]

71. Hills D, Gribi R, Ure J, Buza-Vidas N, Luc S, Jacobsen SE, et al. Hoxb4-YFP reporter mouse
model: a novel tool for tracking HSC development and studying the role of Hoxb4 in
hematopoiesis. Blood. 2011 Mar 31; 117(13):3521–8. [PubMed: 21278354]
72. Ma X, Robin C, Ottersbach K, Dzierzak E. The Ly-6A (Sca-1) GFP transgene is expressed in all
adult mouse hematopoietic stem cells. Stem Cells. 2002; 20(6):514–21. [PubMed: 12456959]
73. Hosen N, Yamane T, Muijtjens M, Pham K, Clarke MF, Weissman IL. Bmi-1-green fluorescent
protein-knock-in mice reveal the dynamic regulation of bmi-1 expression in normal and leukemic
hematopoietic cells. Stem Cells. 2007 Jul; 25(7):1635–44. [PubMed: 17395774]
74. Tadjali M, Zhou S, Rehg J, Sorrentino BP. Prospective isolation of murine hematopoietic stem
cells by expression of an Abcg2/GFP allele. Stem Cells. 2006 Jun; 24(6):1556–63. [PubMed:
16484343]
75. Cairns LA, Moroni E, Levantini E, Giorgetti A, Klinger FG, Ronzoni S, et al. Kit regulatory
elements required for expression in developing hematopoietic and germ cell lineages. Blood. 2003
Author Manuscript

Dec 1; 102(12):3954–62. [PubMed: 12907433]

76. Kimura Y, Ding B, Imai N, Nolan DJ, Butler JM, Rafii S. c-Kit-mediated functional positioning of
stem cells to their niches is essential for maintenance and regeneration of adult hematopoiesis.
PLoS One. 2011; 6(10):e26918. [PubMed: 22046410]
77. Bee T, Swiers G, Muroi S, Pozner A, Nottingham W, Santos AC, et al. Nonredundant roles for
Runx1 alternative promoters reflect their activity at discrete stages of developmental
hematopoiesis. Blood. 2010 Apr 15; 115(15):3042–50. [PubMed: 20139099]
78. Nutt SL, Metcalf D, D'Amico A, Polli M, Wu L. Dynamic regulation of PU.1 expression in
multipotent hematopoietic progenitors. J Exp Med. 2005 Jan 17; 201(2):221–31. [PubMed:
15657291]
79. Zhang J, Varas F, Stadtfeld M, Heck S, Faust N, Graf T. CD41-YFP mice allow in vivo labeling of
megakaryocytic cells and reveal a subset of platelets hyperreactive to thrombin stimulation. Exp
Hematol. 2007 Mar; 35(3):490–9. [PubMed: 17309829]
80. Vassen L, Okayama T, Moroy T. Gfi1b:green fluorescent protein knock-in mice reveal a dynamic
expression pattern of Gfi1b during hematopoiesis that is largely complementary to Gfi1. Blood.
Author Manuscript

2007 Mar 15; 109(6):2356–64. [PubMed: 17095621]

81. Wareing S, Eliades A, Lacaud G, Kouskoff V. ETV2 expression marks blood and endothelium
precursors, including hemogenic endothelium, at the onset of blood development. Dev Dyn. 2012
Sep; 241(9):1454–64. [PubMed: 22733530]
82. Koyano-Nakagawa N, Kweon J, Iacovino M, Shi X, Rasmussen TL, Borges L, et al. Etv2 is
expressed in the yolk sac hematopoietic and endothelial progenitors and regulates Lmo2 gene
expression. Stem Cells. 2012 Aug; 30(8):1611–23. [PubMed: 22628281]
83. Nishimura S, Takahashi S, Kuroha T, Suwabe N, Nagasawa T, Trainor C, et al. A GATA box in
the GATA-1 gene hematopoietic enhancer is a critical element in the network of GATA factors
and sites that regulate this gene. Mol Cell Biol. 2000 Jan; 20(2):713–23. [PubMed: 10611250]
84. Heinrich AC, Pelanda R, Klingmuller U. A mouse model for visualization and conditional
mutations in the erythroid lineage. Blood. 2004 Aug 1; 104(3):659–66. [PubMed: 15090451]
85. Faust N, Varas F, Kelly LM, Heck S, Graf T. Insertion of enhanced green fluorescent protein into
the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood.
Author Manuscript

2000 Jul 15; 96(2):719–26. [PubMed: 10887140]

86. Hamada M, Moriguchi T, Yokomizo T, Morito N, Zhang C, Takahashi S. The mouse mafB 5'-
upstream fragment directs gene expression in myelomonocytic cells, differentiated macrophages
and the ventral spinal cord in transgenic mice. J Biochem. 2003 Aug; 134(2):203–10. [PubMed:
12966068]
87. Sasmono RT, Oceandy D, Pollard JW, Tong W, Pavli P, Wainwright BJ, et al. A macrophage
colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the
mononuclear phagocyte system of the mouse. Blood. 2003 Feb 1; 101(3):1155–63. [PubMed:
12393599]

Methods Mol Biol. Author manuscript; available in PMC 2015 May 04.
 Vacaru et al. Page 20

88. Lohmann F, Bieker JJ. Activation of Eklf expression during hematopoiesis by Gata2 and Smad5
prior to erythroid commitment. Development. 2008 Jun; 135(12):2071–82. [PubMed: 18448565]
Author Manuscript

89. Papadopoulos P, Gutierrez L, van der Linden R, Kong ASJ, Maas A, Drabek D, et al. A dual
reporter mouse model of the human beta-globin locus: applications and limitations. PLoS One.
2012; 7(12):e51272. [PubMed: 23272095]
90. Kissenpfennig A, Henri S, Dubois B, Laplace-Builhe C, Perrin P, Romani N, et al. Dynamics and
function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct
from slower migrating Langerhans cells. Immunity. 2005 May; 22(5):643–54. [PubMed:
15894281]
91. Norris HH, Martin AJ, Lybarger LP, Andersen H, Chervenak DC, Chervenak R. TCRbeta
enhancer activation in early and late lymphoid progenitors. Cell Immunol. 2007 Jun; 247(2):59–
71. [PubMed: 17961527]
92. Singbartl K, Thatte J, Smith ML, Wethmar K, Day K, Ley K. A CD2-green fluorescence protein-
transgenic mouse reveals very late antigen-4-dependent CD8+ lymphocyte rolling in inflamed
venules. J Immunol. 2001 Jun 15; 166(12):7520–6. [PubMed: 11390506]
93. Monroe RJ, Seidl KJ, Gaertner F, Han S, Chen F, Sekiguchi J, et al. RAG2:GFP knockin mice
Author Manuscript

reveal novel aspects of RAG2 expression in primary and peripheral lymphoid tissues. Immunity.
1999 Aug; 11(2):201–12. [PubMed: 10485655]
94. Fontenot JD, Dooley JL, Farr AG, Rudensky AY. Developmental regulation of Foxp3 expression
during ontogeny. J Exp Med. 2005 Oct 3; 202(7):901–6. [PubMed: 16203863]
95. Lochner M, Peduto L, Cherrier M, Sawa S, Langa F, Varona R, et al. In vivo equilibrium of
proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORgamma t+ T cells. J Exp Med. 2008
Jun 9; 205(6):1381–93. [PubMed: 18504307]
96. Fuxa M, Busslinger M. Reporter gene insertions reveal a strictly B lymphoid-specific expression
pattern of Pax5 in support of its B cell identity function. J Immunol. 2007 Jun 15; 178(12):8222–8.
[PubMed: 17600970]
97. Kuwata N, Igarashi H, Ohmura T, Aizawa S, Sakaguchi N. Cutting edge: absence of expression of
RAG1 in peritoneal B-1 cells detected by knocking into RAG1 locus with green fluorescent
protein gene. J Immunol. 1999 Dec 15; 163(12):6355–9. [PubMed: 10586023]
98. Kallies A, Hasbold J, Tarlinton DM, Dietrich W, Corcoran LM, Hodgkin PD, et al. Plasma cell
ontogeny defined by quantitative changes in blimp-1 expression. J Exp Med. 2004 Oct 18; 200(8):
Author Manuscript

967–77. [PubMed: 15492122]

99. Jung S, Aliberti J, Graemmel P, Sunshine MJ, Kreutzberg GW, Sher A, et al. Analysis of
fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter
gene insertion. Mol Cell Biol. 2000 Jun; 20(11):4106–14. [PubMed: 10805752]
100. Yang J, Hills D, Taylor E, Pfeffer K, Ure J, Medvinsky A. Transgenic tools for analysis of the
haematopoietic system: knock-in CD45 reporter and deletor mice. J Immunol Methods. 2008 Sep
15; 337(2):81–7. [PubMed: 18602924]
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Figure 1. Basic design of a fluorescent transgenic construct

"R" marks the restriction enzyme sites used for removal of the bacterial backbone before
transgene injection. s/pA represents the splicing and polyadenylation signals. A stop codon
(denoted by "X") should also be included in the construct design. The enhancer may be
positioned upstream or downstream from the promoter and sequences encoding the FP and
regulatory signals. For additional details, see text.
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Figure 2. Basic steps in the generation of a fluorescent transgenic reporter mouse line
Pronuclear injection of the transgene is shown in this cartoon but transgenic mouse lines can
also be generated by blastocyst injection or embryo aggregation with genetically modified
ES cells (see text).
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Figure 3. Tagging of primitive and definitive erythroid lineages using GFP reporters
(A) GFP expression in the primitive erythroid cells of an embryonic stage (E)8.5 ε-globin-
H2B-GFP embryo (24) (left panel, whole embryo, scale bar 200 μm; right panel, magnified
view of yolk sac, scale bar, 50μm). Embryos were photographed on a Zeiss Lumar V12
stereomicroscope equipped with epifluorescence illumination and a NeoLumar S 1.5X FWD
30 mm objective. (B) Wet preparation of green fluorescent erythroid cells from the bone
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marrow (BM) and peripheral blood (PB) of an adult miR-144/451-GFP knock-in mouse
(26). The cells were photographed on a Zeiss Axio Observer Z1 inverted microscope with
epifluorescence illumination and a Plan-Apochromat 20X/0.8 objective. Scale bar, 20μm.
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Table 1

Transgenic mouse lines expressing fluorescent reporter proteins in the hematopoietic system.
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Gene Reporter Lineage labeled Reference

Gata2 GFP (Ki) HSC (70)

Hoxb4 YFP (Ki) HSC (71)

Ly-6A GFP (Tg) HSC (72)

Bmi1 GFP (Ki) HSC§ (73)

Abcg2 IRES-GFP (Ki) HSC, erythroid (74)

c-Kit GFP (Tg) HSC, progenitors (75)

c-Kit GFP (CKO) HSC, progenitors (76)

Runx1 GFP (Tg) HSC, progenitors (77)

Pu.1 IRES-GFP (Ki) HSC, lymphoid and myeloid progenitors (78)

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CD41 farnesyl-YFP (Tg) HSC, progenitors, megakaryocytes, platelets (79)

Gfi1B GFP (Ki) HSC, erythroid and myeloid progenitors (80)

Etv2 GFP (Tg) Hematopoietic and endothelial progenitors (81)

Etv2 EYFP (Tg) Hematopoietic and endothelial progenitors (82)

Gata1 GFP (Tg) Hemangioblast, EryP, EryD, megakaryocytes (83)

EpoR GFP-Cre (Ki) Erythroid progenitors, endothelial (84)

Lysozyme M EGFP (Ki) Myelomonocytic cells, including macrophages and granulocytes (85)

MafB GFP (Tg) Myelomonocytic lineages of hematopoietic cells, peritoneal macrophages (86)

c-fms EGFP (Tg) Macrophages, dendritic cells, myeloid cells (87)

β-globin ECFP (Tg) MEP, EryD (50)

miR-144/451 EGFP (Ki) EryD (26)

Eklf GFP (Tg) EryD (88)

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ε-globin KGFP (Tg); H2B- GFP (Tg) EryP (23, 24)

γ-globin EGFP EryP (89)

β-globin DsRed EryD

Langerin IRES-EGFP (Ki) Langerhans cells (90)

TCRb GFP (Tg) Lymphoid progenitors (91)

Runx1 IRES-GFP (Ki) Lymphoid, myeloid, lower levels in erythroid (29)

CD2 EGFP (Tg) T lymphoid cells (92)

Rag2 GFP (Ki) T and B lymphoid cells (93)

FoxP3 GFP (Ki) T regulatory lymphoid cells (94)

Ror(gT) EGFP (Ki) T helper 17 lymphoid cells (95)

Pax5 EGFP (Ki) Pre-B, B lymphoid cells (96)

Rag1 GFP (Ki) B lymphoid cells (97)

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Blimp1 IRES-EGFP (Ki) B lymphoid cells, plasma cells (98)

CX3CR1 GFP (Ki) Macrophages, monocytes, NK cells, dendritic cells, microglia (99)

CD45 YFP (Ki) Widespread hematopoietic (100)

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Abbreviations: Tg, Transgenic; KI, Knock-in; CKO, Conditional Knock-out; GFP, Green fluorescent protein; IRES, Internal ribosomal entry site;
EGFP, enhanced GFP; YFP, Yellow Fluorescent Protein; CFP, Cyan Fluorescent Protein; HSC, hematopoietic stem cell; EryP, primitive erythroid;
EryD, definitive erythroid; MEP, Megakaryocyte-erythroid progenitor; NK, Natural Killer cells.
§
GFP expression is highest in hematopoietic stem cells (HSCs) and is downregulated during lineage commitment and differentiation.
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Table 2

PCR primer pair sequences for commonly used fluorescent reporters

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Reporter Primer Sequences Tm Product size

GFP Forward - 5’-CAT GAG CAA GGG CGA GGA ACT-3’ 55°C 750 bp
Reverse - 5’-CAG CAG CGG TCA CAA ACT CC -3'

ExFP (for GFP, CFP,YFP) Forward - 5'-CAC CAT CTT CTT CAA GGA CGA C-3' 53°C 350 bp
Reverse - 5'-TTC TCG TTG GGG TCT TTG C-3'

mCherry Forward -5’-GAT ACT CGA CC AAG CAA GGG CGA GG-3’ 53°C 720 bp
Reverse -5’-CATA ACT CGA GTT ATG TAC AGC TCG TC-3’

tdTomato Forward -5’-ATG GAG GGC TCC ATG AAC G -3’ 50°C 370 bp
Reverse - 5’-CCC ATG GTC TTC TTC TGC -3’

Abbreviations: bp, base pairs; Tm, annealing temperature

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