Name: lineage tracing of inducible fluorescently-labeled stem cells in the adult mouse brain
Uploaded: 2022-05-20T14:00:00
Description: Watch this Scientific Journal Video about Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain at JoVE.com

The authors wish to thank Dr. Diana Carlone (Boston Children&#39;s Hospital, Harvard Medical School) and Dr. Matthew Lynes (Joslin Diabetes Center; Maine Medical Center Research Institute) for guidance utilizing mTert-rtTA animals.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of The Ohio State University.
1. Creation of a lineage tracing Tet-On mouse line, breeding strategies, and genotyping for experimental cohort mice
NOTE: Here, we outline creation of a Tet-On mouse system with Rosa-mTmG as the fluorescent reporter gene that undergoes Cre recombination, however various other Cre-recombination-driven reporter lines ca...

A method for the creation, utilization, and analysis of a triple transgenic lineage tracing mouse line marking stem cells within the adult mouse brain in vivo is described. When combined with immunostaining or brain clearing, the identification and characterization of traced fluorescent cells across the brain can be accomplished. This technique offers the ability to understand the plastic/regenerative/remodeling potential of labeled stem cells as they activate, migrate, differentiate, or proliferate. While previ...

Value of a lineage tracing mouse line 
Analysis of stem cells in vivo can be difficult since many assays that examine such cells only focus on characterizing these cells at the time of death of the animal, which represents a terminal snapshot in time. To better understand the processes of proliferation, differentiation, and migration of progenitor, intermediate/transition cell types, and mature cells over time, a longitudinal analysis approach is required. This can be achieved with lineage tracing studies whereby stem/progenitor cells are marked indelibly and can be followed for any length of time afterwards1.
In the adult mammalian brain, the process of neurogenesis by which adult-born neurons are created from stem and progenitor cells was first analyzed via label retention with thymidine-H3&#160;2,3,4 or 5-bromo-2&#39;-deoxyuridine (BrdU)5,6,7,8. In these studies, proliferative cells were marked with the thymine analog, which was incorporated into the cells&#39; DNA during replication and cell division/proliferation. The marked cell, as well as their progeny, therefore contained this analog, which were then identified post-mortem. However, while thymidine-H3 and BrdU allowed for the marking of proliferative cells and their progeny in brain regions where stem cells were poorly understood, these studies were hindered by the downsides of these tools. Thymidine-H3 induces cell-cycle arrest, apoptosis, and dose-dependent DNA synthesis inhibition9, while BrdU marks cells at varying levels depending on the route of administration10 and is taken up by cells during repair or apoptosis, as well as during cell division11. More efficient labeling methods have been utilized recently, such as labeling with 5-ethynyl-2&#39;-deoxyuridine (EdU), but many of the same issues as BrdU still remain12. These approaches are also limiting in that they mark any proliferative cell, not just stem cells, and thus interpretation of results can be confounded. In the neurogenic lineage, all stem and progenitor cells retain mitotic capacity, and only terminal/mature neurons are not proliferative. For astrocytes and microglia, but not oligodendrocytes, proliferation is maintained indefinitely13,14,15.
Transgenic mice used to lineage trace only cells that express a protein of interest, such as one confirmed to identify adult stem cells as we use here, have therefore become more common when investigating stem cells and their progeny. While difficult to generate through mouse transgenic approaches, lineage-tracing mouse lines allow for the tracing of specifically marked cells in the brain, and do not rely on proliferation alone. In the Tet-On transgenic mouse system, administration of tetracycline or doxycycline (a tetracycline derivative) induces Cre-recombinase expression in cells that have been engineered with a reverse tetracycline transactivator (rtTA), which is transcribed by a promoter of interest. The Tet-inducible Cre-driven recombination will then activate the expression of an indelible fluorescent or luminescent protein in the cells of interest, depending on the Rosa-reporter mouse employed. These indelibly marked cells continue to express this reporter after division, differentiation, or migration, allowing the tracking of these cells and their progeny over time or after different interventions16. Advantages of transgenic lineage-tracing approaches include: 1) specificity of tracing to a particular cell lineage or progenitor/stem cell marked by the rtTA, 2) indelible expression of the fluorescent or luminescent protein despite cell turnover or differentiation, 3) low toxicity, 4) conditional activation during any point in the animal&#39;s life cycle, and 5) ease of use with common assays, including immunostaining/immunofluorescence16.
Other methods of temporally inducible reporter tools in mice include the use of Cre-ERT2 mice, which can be paired with ROSA-GFP, ROSA-mTmG, or other fluorescent reporter transgenes. In these animals, a cell-specific regulatory element, such as a promoter or enhancer region of interest, drives the production of Cre recombinase, which can only be activated via the administration of tamoxifen. While Cre-ERT2 mouse lines allow for the induction of Cre in specific cell lines, there is a wealth of knowledge detailing the effects of tamoxifen on adult neurogenesis17,18. Additionally, there exists many ROSA-driven fluorescent reporter genes that can be utilized instead of GFP or mTmG, including YFP or CFP, which would allow for alternate fluorescent labeling in other fluorescent wavelengths. These fluorescent reporter genes can be used with Tet-On or Cre-ERT2 systems.
Telomerase reverse transcriptase (TERT) is the rate-limiting component of the holoenzyme telomerase, which works to extend telomeres after they are shortened during cell division19,20. TERT has been identified as a marker of adult tissue stem cells in the intestine21,22, bone marrow21, liver23, adipose24, endometrium25,26, and bone1. Whether TERT expression in these adult stem cells is solely for telomerase-extending activity or to perform non-canonical TERT roles27 is still unknown. TERT-expressing quiescent adult stem cells (qASCs) have been identified and traced throughout the body with transgenic lineage tracing mice to study the multipotency and self-renewal ability of these stem cells, as well as their potential to activate, proliferate, differentiate, and migrate1,22,23,28. The creation of the Tert-rtTA transgene has been performed previously1. In this paper, we will describe the use of a lineage tracing TERT mouse line to study TERT + qASCs which we have identified as a novel ASC population in the adult mouse brain.
Generation of a transgenic lineage tracing mouse line 
To generate a lineage-tracing mouse line using the Tet-On system, three transgenes must be combined within a single animal through mouse mating. The first is a rtTA, expressed under the control of the promoter of the gene of interest (ours is TERT-rtTA). In a cell that expresses this gene of interest, the rtTA will therefore be expressed. The second is an oTet-Cre gene, which contains a tetracycline response element (TRE) that will allow for the transcription of Cre recombinase in the presence of both a rtTA fusion transcript and tetracycline or doxycycline. Tetracycline or doxycycline can be administered to an animal via drinking water or chow29. Finally, there must be a gene that will be activated by Cre recombinase cleavage. In this manuscript, the labeling gene we will describe is the Rosa26-mTmG gene, which until Cre recombination will ubiquitously transcribe mTomato (membrane red fluorescence in all cells). However, if a cell expresses the rtTA transcript and contains tetracycline or doxycycline, Cre recombination of a set of lox P sites between the mTomato and mGFP sites will change the R26 site and cause the cell to produce membrane EGFP (mGFP, or membrane green fluorescence) instead of membrane tomato. The indelible nature of this mGFP signal will allow for in vivo labeling and tracing of cells as they proliferate, migrate, and differentiate. Following the trace, cells can be analyzed for expression of GFP via immunofluorescence in standard cryosections or thicker optically cleared brains.
In this paper, we will describe the use of the specific mouse line, mTert-rtTA::oTet-Cre::Rosa-mTmG. To create this triple transgenic mouse line, we first mated oTet-Cre animals (The Jackson Lab strain #006234) to Rosa-mTmG animals (The Jackson Lab strain #007676) to create oTet-Cre::Rosa-mTmG double transgenic mice. These animals were genotyped with primers and PCR templates indicated in Supplementary Tables 1 and 2. mTert-rtTA mice (created by David Breault1) were mated to oTet-Cre::Rosa-mTmG animals to create mTert-rtTA::oTet-Cre::Rosa-mTmG mice (Figure 1A)1,30. The mechanism of action of the mTert-rtTA::oTet-Cre::Rosa-mTmG mouse line is illustrated in Figure 1B.
Doxycycline induction and pulse-chase design 
When planning experiments, it is important to consider several factors that will affect the outcome of lineage tracing experiments, including the age of the animal at doxycycline induction, the length of doxycycline administration (the &#34;pulse&#34; period), the length of time after doxycycline removal before tissue collection (the &#34;chase&#34; period), and the timing of any interventions during these processes. These study design considerations are essential for understanding the labeled cells and their progeny at the conclusion of the experiment. The first step in the process is to identify the age of interest of the animal and the length of the doxycycline administration. Longer pulse periods will allow for the potential for more cells to express the rtTA-linked promoter to be expressed and more cells of interest to be indelibly marked. A cell type that exhibits transient expression of the gene of interest may require a longer pulse period than cells that continuously express the gene of interest. However, if the goal of the study is to understand short-term effects of the cell of interest or an acute intervention, the pulse period cannot be too long, as once a cell is marked, it will be traced through any number of changes during the remainder of the pulse period. In some of our studies, a 2 day pulse with no chase period was used to more closely mimic a direct-reporter for TERT. This is because the minimum length of time for recombination of the Rosa-mTmG gene is 2 days31.
The next essential period in a pulse-chase experiment is the length between removal of doxycycline and perfusion of the animal, also known as the &#39;chase&#39;. The half-life of doxycycline in mice is approximately 170 minutes regardless of administration route, allowing for the conclusion that doxycycline induction is likely no longer occurring several hours after removal32. However, it is important to note that doxycycline metabolism is slower in aged mice, which may lead to longer effective &#39;pulse&#39; periods than young animals33.
During the chase period, labeled cells will continue to be labeled, even after proliferation, differentiation, or migration. The progeny of these cells will also be labeled. The goal of the study will shape the length of the pulse-chase paradigm. If the goal of the study is to understand the regeneration of a tissue over a long period of time with the cells of interest, a long chase period may be required. Finally, the timing of any interventions or treatments must be decided. Administration during the pulse period will affect the cells expressing the gene of interest as well as any progeny created during this time, whereas administration during the chase period may affect mostly progeny of the cells of interest, although this will depend on how quickly the labeled cells divide or differentiate. Examples of pulse-chase experimental designs we have utilized are outlined in Figure 2A.
It is also important to be aware of the possibility of background GFP signal due to leaky expression. Leaky expression occurs as a result of inherent binding potential between rtTA and the Otet sequences in the absence of doxycycline, and residual activity of the Otet transgene in the absence of rtTA (reviewed in34). Leaky expression represents one weakness of the Tet systems, and although the leak is often an acceptable downside to the system, situations where the leaky expression can lead to toxicity and mortality, such as with diptheria toxin (DTA) in Otet-DTA animals can limit the use of these systems35.
Brain processing, sectioning, and immunofluorescence of thin sections for microscopy 
To analyze the brains of mice after a lineage tracing experiment, mice must first be perfused via transcardial perfusion to remove blood and CSF that can lead to high autofluorescence in the brain. There are two commonly used fixatives that the animal may be perfused with: Histochoice Tissue Fixative, a glyoxal fixative (GF), or 4% paraformaldehyde (PFA). Glyoxal fixatives allow for a relatively gentle fixation process with less cross-linking than PFA. This reduces tissue stiffness, reduces the need for antigen retrieval, and allows for less concentrated antibody solutions during immunostaining. However, if they contain methanol this may serve to increase autofluorescence36. On the other hand, PFA will often allow for a more robust fixation, and while antigen retrieval will be required during immunostaining, there are antibodies that will only work with PFA-fixed tissues, and perfusion with 4% PFA is required for the optical clearing technique described later.
Following perfusion is a post-fixation step, in which brains are immersed in the same type of fixative utilized during the perfusion overnight. This allows for any brain regions that may not have been fully fixed during the perfusion to continue to fix. Next, brains will be incubated in sucrose in phosphate buffered saline (PBS) to remove as much water as possible before the freezing step to prevent ice formation within the tissue (&#34;cryo-preservation&#34;). Brains may then be cut into any number of sagittal, coronal, or transverse tissue sections for processing. For both precision and reproducibility, calibrated brain blocks need to be used in a way that ensures consistent bregma cuts between animals. The most important factor that can affect how brains are sectioned is the brain region(s) of interest. For example, while a sagittal cut may allow for more brain regions to be analyzed in one tissue section, this cut will not allow analysis of neuro/gliogenic regions of the ventricular-subventricular zone (V-SVZ) as the lateral and medial sides of the lateral ventricle will be impossible to discern in that dimension and are better observed in the coronal plane. This can be an important distinction to make in adult neurogenesis studies, as the lateral side of the lateral ventricle contains the neurogenic V-SVZ, while the medial wall of the lateral ventricle is a more gliogenic niche37.
After tissues have been divided into coronal, sagittal, or transverse sections, they will be frozen into blocks using Optimal Cooling Temperature (OCT) embedding solution. Here, brains are frozen within this embedding material, with the use of a mixture of dry ice and ethanol. If thin section analysis is required, blocks will be sliced on a cryostat, and depending on the downstream analysis required can be adhered to positively charged glass slides as thin sections (&#60;20 &#181;m). Glass slides that are not charged can also be used, but the use of uncharged slides may result in tissues becoming unstuck from the slides38. If medium-thickness free-floating tissue sections (&#62;20 &#181;m) are required, tissues will be collected as free-floating sections. If thick sections (0.5-4 mm) are required, slicing non-frozen brain sections with a vibratome is required. It is important to note the storage differences between sections on slides, which require freezing at -20 to -80 &#176;C and free-floating sections, which will be stored at 4 &#176;C.
Brain clearing and whole mount immunofluorescent confocal microscopy 
If a broader, yet less detailed analysis of the lineage-traced cells in the mouse brain is desired, a comprehensive analysis of thicker segments of brain tissue is possible with iDISCO brain clearing39 followed by immunostaining and tiled z-stack confocal microscopy or light-sheet microscopy. Here, large sections of mouse brain will be optically cleared (a process of delipidation39), which better allows for fluorescent signal imaging across a 1 mm tissue section. The downsides of this process are high autofluorescence and a diminished ability to obtain high resolution images of cell morphology and detailed co-staining analysis due to the increased working distances required when compared to more traditional methods (thin cryosections or free-floating sections). For this reason, we recommend brain clearing to analyze the large-scale lineage tracing results throughout multiple brain areas, instead of attempting to characterize or analyze individual cells.

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			<td>Antibody diluent</td>
			<td>Agilent</td>
			<td>S080983-2</td>
			<td></td>
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			<td>Antigen Retrieval Solution</td>
			<td>Agilent</td>
			<td>S2367</td>
			<td></td>
		</tr>
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			<td>anti-GFP antibody</td>
			<td>Invitrogen</td>
			<td>A2311</td>
			<td>Use at 1:500-1:000</td>
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			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A18-4</td>
			<td></td>
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			<td>B6.Cg-Tg(tet0-cre)1Jaw/J</td>
			<td>The Jackson Laboratory</td>
			<td>JAX:006234</td>
			<td></td>
		</tr>
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			<td>B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J</td>
			<td>The Jackson Laboratory</td>
			<td>JAX:007676</td>
			<td></td>
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			<td>Blocking Solution (IHC)</td>
			<td>Millipore Sigma</td>
			<td>20773</td>
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			<td>Blunt needle</td>
			<td>BSTEAN</td>
			<td>X0012SYHIV</td>
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			<td>Coronal brain block</td>
			<td>Braintree</td>
			<td>BS-2000C</td>
			<td></td>
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			<td>Cover slip</td>
			<td>Corning</td>
			<td>2850-22</td>
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			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1900</td>
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			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D564</td>
			<td></td>
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			<td>Dibenzyl ether</td>
			<td>Millipore Sigma</td>
			<td>33630</td>
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			<td>Dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td></td>
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			<td>Dimethyl Sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
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			<td>Donkey Serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
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			<td>Doxycycline Hyclate</td>
			<td>Sigma-Aldrich</td>
			<td>D9891</td>
			<td></td>
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			<td>Glycine</td>
			<td>Bio-Rad</td>
			<td>161-0718</td>
			<td></td>
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			<td>Heparin Sodium Salt from Porcine Mucosa</td>
			<td>Sigma-Aldrich</td>
			<td>H3393</td>
			<td></td>
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			<td>Histochoice Molecular Biology Tissue Fixative</td>
			<td>Amresco</td>
			<td>H120</td>
			<td>This product has since been discontinued, but can be replaced with Glyo-Fixx (Thermo Scientific, 6764265)</td>
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			<td>Hydrogen Peroxide Solution</td>
			<td>Sigma-Aldrich</td>
			<td>516813</td>
			<td></td>
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			<td>IHC Select TBS Rinse Buffer</td>
			<td>Millipore Sigma</td>
			<td>20845</td>
			<td></td>
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			<td>Ketamine</td>
			<td>Westward</td>
			<td>0143-95095-10</td>
			<td>This product requires a DEA license for storage and use.</td>
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			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452-4</td>
			<td></td>
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			<td>Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
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			<td>Milipore Block</td>
			<td>Millipore</td>
			<td>20773</td>
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			<td>Mounting medium</td>
			<td>Millipore Sigma</td>
			<td>5013</td>
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			<td>Optimal Cutting Temperature Solution</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
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			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
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			<td>PBS Solution (10X)</td>
			<td>Teknova</td>
			<td>P0496</td>
			<td></td>
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			<td>Sagittal brain block</td>
			<td>Braintree</td>
			<td>RBM-2000S</td>
			<td></td>
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			<td>Saline</td>
			<td>Braun</td>
			<td>S8004-5264</td>
			<td></td>
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			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td></td>
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			<td>Sudan (Typogen) Black</td>
			<td>Millipore Sigma</td>
			<td>199664</td>
			<td></td>
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			<td>Tetrahydrofuran</td>
			<td>Sigma-Aldrich</td>
			<td>186562</td>
			<td></td>
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			<td>Tissue cartridge</td>
			<td>Simport</td>
			<td>M512</td>
			<td></td>
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			<td>Triton X-100</td>
			<td>Bio-Rad</td>
			<td>1610407</td>
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			<td>Tween-20</td>
			<td>Millipore Sigma</td>
			<td>655205</td>
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			<td>Xylazine</td>
			<td>AnaSed</td>
			<td>sc-362950Rx</td>
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lineage tracing of inducible fluorescently-labeled stem cells in the adult mouse brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

While the fluorescent signal resulting from a Tet-On system with a fluorescent reporter will vary depending on the promoter of interest and fluorophore(s) used, we will describe how findings were analyzed with mTert-rtTA::oTet-Cre::Rosa-mTmG animals. Analysis of adult brains after a pulse-chase resulted in membrane GFP-expressing cells throughout various anatomical niches. The difference in fluorescent intensity between various cell types must be noted. One variable regarding fluorescent intensity is the size of the cell...

Watch this Scientific Journal Video about Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain at JoVE.com

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

Marking stem cells and their progeny with an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation of adult stem cells in the brain in vivo. Lineage tracing can reveal novel information about lineage commitment, response to interventions, and multi potency. Advantages of transgenic lineage tracing approaches include specificity of tracing a particular cell lineage, indelible expression of the fluorescent protein regardless of cell turnover or differentiation, low toxicity of doxycyline, conditional activation, and ease of use with common downstream assays on lineage trace tissues including immunostaining and immunofluorescence.Due to the broad implication of plasticity and cell in the adult brain, the characterization and analysis of adult stem cells will help inform of the study of neurodegenerative diseases, metabolic impacts on brain plasticity, aging, and other related conditions. To begin, warm slides to room temperature and post-fix with ice cold acetone for 15 minutes. Wash slides for five minutes in 1X rinse buffer, shaking at 60 rotations per minute at room temperature between each step.Permeabilize for standing of nuclear antigens with 0.3%Triton X-100 for 10 minutes at room temperature. Permeabilize for standing of cytoplasmic antigens with 0.3%Tween-20 for 10 minutes at room temperature. Perform antigen retrieval by microwaving slides in 50 to 100 milliliters of 1x DAKO Antigen Retrieval Solution on low for 10 minutes, twice.Next, rinse with buffer for five minutes at room temperature. incubate slides in 0.3%Typogen Black in 70%ethanol for 20 minutes at room temperature and then wash with rinse buffer. Draw hydrophobic barrier around tissue.Block for 20 minutes at 37 degrees Celsius with blocking reagent. And incubate with primary antibody diluted in antibody diluent overnight at four degree Celsius. Wash slides for 10 minutes at room temperature.The next day, incubate sections in Alexa Fluor secondary antibodies for 10 minutes at room temperature. Then wash slides twice in rinse buffer for 10 minutes. Cover brain sections in 100 microliters of one to 500 anti-GFP antibody conjugated to AlexaFluor 488, and incubate overnight at four degrees Celsius to boost the endogenous Fluor4, marking the traced cells.The next day, wash with rinse buffer two times. And then wash in running DI water for five minutes. Counterstain with 100 nanograms per milliliter 4'6-diamidino-2-phenylindole for five minutes.Then wash in running DI water for five minutes. Add a drop of fluorescent safe mounting medium on top of each tissue section and seal with a 22 millimeter by 22 millimeter coverslip. Imaging is recommended on the day of mounting to ensure optimal signal.To begin fixation and sectioning following transcardial perfusion post-fix brains in 4%PFA overnight at four degrees Celsius. And then again for one hour at room temperature. Wash brains in 1x PBS for an hour twice at room temperature.Cut the brain into one millimeter thick sections using the sagittal brain block and place into five milliliter micro centrifuge tubes containing 1x PBS. For methanol pre-treatment, start by washing with 1x PBS for one hour twice at room temperature. Wash in 50%methanol for one hour at room temperature.Wash in 80%methanol for one hour at room temperature. Wash in 100%methanol for one hour, twice, at room temperature. Bleach samples with 5%hydrogen peroxide in 20%dimethyl sulfoxide and methanol at four degrees Celsius overnight.After bleaching, wash samples in methanol for one hour twice at room temperature. Wash in 20%dimethyl sulfoxide and methanol for one hour twice at room temperature. Wash in 80%methanol for one hour at room temperature.Wash in 50%methanol for one hour at room temperature. Wash in PBS for one hour twice at room temperature. Wash in PBS 0.2%Triton X-100 for one hour twice at room temperature.For immunostain of clearing brains, begin by incubating samples in 1x PBS, 0.2%Triton X-100, 20%DMSO, 0.3 molar glycine, at 37 degrees Celsius overnight on an orbital shaker. Block in 1x PBS, 0.2%Triton X-100, 10%DMSO, 6%goat serum at 37 degrees Celsius for three days on an orbital shaker. Wash samples in 1x PBS, 0.2%Tween-20, 10 micrograms per milliliters heparin sodium salt from porcine mucosa for one hour twice at 37 degrees Celsius.Next, incubate in 1x PBS, 0.2%Tween-20, 10 micrograms per milliliters heparin, 5%DMSO, 3%goat serum containing one to 500 concentration of rabbit anti GFP AlexaFluor 488 at 37 degrees Celsius on an orbital shaker for two days. Wash samples in 1x PBS, 0.2%Tween-20 with 10 micrograms per milliliter heparin for one hour at 37 degrees Celsius on an orbital shaker three times and then once a day for two days. Incubate samples overnight in one milliliter of 50%Tetrahydrofuran and water.Incubate samples for one hour in one milliliter of 80%THF Tetrahydrofuran and water. Incubate samples twice for one hour in 100%Tetrahydrofuran. Dry samples with a sterile wipe and incubate in dichloromethane until they sink to the bottom of the vial.Do not incubate for more than 60 minutes. Incubate samples in one milliliter of dibenzyl ether until clear. Store samples in dibenzyl ether at room temperature until ready to image.To image immunostained brain sections, analyze slides via confocal microscopy, where co-expression of multiple antibodies can be confirmed. We use a Leica Stellaris five microscope with HyD S hybrid detectors. To image cleared brains, use an inverted confocal microscope with an automated stage and automated tiling function.Start by laying a cleared brain section flat against a glass bottom dish in submerge in dibenzyl ether. A noticeable difference in fluorescent intensity across different brain regions and various cell types was observed following a pulse chase period in the lineage traced brains. Choroid epithelial cells in the choroid plexus had a thick cell membrane and strong green fluorescent signal indicating they derived from Tert positive cells which was easily distinguishable from the surrounding cells.The other smaller cell types in the choroid plexus stroma had a weaker GFP signal. In the cerebellum, Bergmann glial cells expressed higher levels of GFP than basket cells, and variation in GFP expression also existed between individual basket cells. For factory sensory neuron axons within the glomerular layer of the olfactory bulb also expressed high levels of GFP Olfactory sensory neuron axons that fill the glomerular layer of the olfactory bulb expressed high levels of membrane tomato, or red fluorescence when compared to most other brain regions even when the same neurons are GFP positive indicating that some cells appeared to lose mTomato signal more slowly during lineage tracing.In contrast, in the choroid plexus GFP positive cells expressed low mTomato. In most other brain regions, tomato signals expressed at low level across most cell types with endothelial cells being some of the only cells whose fluorescent signal was bright enough to stand out distinctly from the red fluorescent background of the brain tissue. The most important thing to remember is that the fluorescent signal in cleared brains fades faster than most other immuno assays.Remember to image your samples as soon as possible and within seven days. Following, imaging of the brain slices or clear brains, colorization of markers with the GFP signals and our quantification of Fluorence can be undertaken using software programs.

Research

JoVE Journal

Neuroscience

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...