We would like to thank David R. Rodriguez, M.A. for the graphic design and photography in this manuscript. This work was supported by funding from the GCCRI, the NIH/NIA (5R21AG033339) and the Cancer Center Support Grant (P30CA054174) to the UTHSCSA Flow Cytometry Core facility and the UTHSCSA Advanced Nucleic Acids Core Facility.

The authors declare&#160;no competing financial interests.

The in vivo mutagenesis assay described herein is based on the LacI transgenic mouse model originally generated by Kohler et al.18 This model utilizes a &#955; phage vector carrying a lacI reporter gene. The two cos sites flanking the vector allow for a relatively simple recovery and subsequent packaging into infectious phage particles, used to infect E. coli. A blue plaque will be generated by phage-infected E. coli that contain a mutated LacI...

In most tissues, differentiated cells have a limited life-span. To maintain functional integrity, long-lived, tissue-specific stem cells continuously produce progenitor cells that in turn give rise to the fully differentiated cells required for the function of that particular tissue. Stem cells also replenish their own compartment through a process called self-renewal. Thus, stem cells are responsible for maintaining the functional integrity of the tissue they reside in. Therefore, it is imperative that they are equipped with robust mechanisms to sense and potentially repair damaged DNA. If not, they may acquire multiple genomic (potentially harmful) perturbations, which can be inherited by their progeny. Understanding how stem cells safe-guard their genome during the life span of an organism is an important question and may help us understand why genomic instability is linked with cancer and some other age-related diseases (reviewed in1,2).
Controlling genomic integrity of a tissue at the level of stem cells or early progenitor cell populations can be achieved by either eliminating defective stem (or progenitor) cells via cell death, senescence or differentiation, and/or by efficient repair of damaged DNA. Recent studies have demonstrated that it is possible to measure certain types of DNA repair directly in these rare populations3-6. It was found that, for example in the hematopoietic system, double strand DNA breaks can be repaired by homologous recombination (HR) or non-homologous end joining (NHEJ), the latter being a repair process of lower fidelity and thus increased risk of making errors. Both are being utilized in hematopoietic stem cells (HSCs)4,5, however, in mice it seems it is predominantly NHEJ in HSCs whereas early progenitors cells utilize HR4. A similar observation was made for stem cells in the skin6. Interestingly, in human HSCs HR, not NHEJ, seems to be the repair mechanism of choice for double strand breaks3. Whether this functional difference between the two species is real or merely represents a technological or experimental difference remains to be seen.
A stem cell&#39;s repertoire to repair damaged DNA is likely to include other DNA repair mechanisms, such as base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR). BER and NER are responsible for repairing single or multiple base pair lesions in single stranded DNA, while MMR fixes base-base mismatches and insertion/deletion loops; these types of DNA damage cannot be repaired by NHEJ or HR. Supporting this notion are several studies from the hematopoietic system demonstrating a link between alterations in one of these pathways and abnormalities in the HSC compartment7-9, as well as an increased risk of developing myelodysplastic syndrome10-16, a disease that originates in the HSC and that is associated with increasing genomic instability as the disease progresses17. As of yet, measurements of BER, NER, and MMR directly in HSCs have not been reported.
In addition to elucidating the various processes that control tissue integrity at a mechanistic level, it is imperative to be able to measure the extent of mutated DNA, so that the consequences of aberrations in one of these processes can be tested, e.g. in normal versus genetically engineered stem cells or in old versus young. However, the development of a relevant assay is difficult because of the paucity of tissue-specific stem cells and the lack of culture conditions that preserves &#34;stemness&#34;. Moreover, such an assay should be amendable to environmental and genetic manipulations. A possible solution to these limitations and requirements is the use of mouse models that are specifically engineered to detect DNA mutations.
Multiple transgenic mouse models for mutation detection have been developed. For example, LacI transgenic mice18 carry a recoverable &#955; phage vector encoding the LacI reporter system, in which the LacI gene encodes a suppressor of the Lac operator and serves as the mutation reporter. Upon mutation of the LacI gene, the Lac operator is activated and &#946;-galactosidase is produced. &#946;-galactosidase cleaves the chromogenic substrate X-gal (5-bromo-4-chloro-e-indolyl-&#946;-D-galactopyranoside), which turns it blue. The cos sites flanking the LacI vector allows easy recovery by lambda phage proteins and subsequent infection of E. coli. After incubating infected E. coli on agarose that contains the X-gal substrate, plaques can be scored. Blue plaques contain a putative mutant Lac-I carrying phage, while clear plaques harbor non-mutants. The frequency of blue plaques (among the clear ones) indicates the mutant frequency in the original cell population the DNA was extracted from. Moreover, the &#955; phage hosting the LacI target can be readily sequenced using PCR techniques for relatively high throughput analysis. Sequencing multiple mutant LacI genes will reveal important information about the mutation spectrum, which in turn may point to possible deficiencies in specific DNA repair pathways or to specific genotoxic events. The LacI transgenic system has been standardized across multiple laboratories19 and the reagents are available commercially. One major disadvantage of the LacI system is the limited ability to detect large deletions or rearrangements; therefore, other methods, e.g. multi-color FISH on metaphase spreads need to be used to compliment this deficiency.
Within the &#955; phage vector of the LacI mouse model, there is a much smaller gene, CII, available for mutation analysis. Its size and the fact that mutants can be selected makes this a less labor-intensive and cheaper assay20 than the LacI gene analysis. However, the LacI gene is more extensively studied for mutagenesis21 and the sensitivity of the gene to mutations has been well characterized so that there is a clear understanding of the amino acid residues that generate a phenotypic response on a chromogenic substrate22-25.
Other mouse models for mutation detection include the use of the &#934;X174 or the LacZ transgenes. The &#934;X174 transgenic mouse model, with the original A:T&#8594;G:C reversion mutation assay26 or the forward mutation assay27 that allows detection of a spectrum of base pair substitutions, represents a less costly system than the LacI model. However, the mutational screen in the forward assay is not trivial and the mutation spectrum of the &#934;X174 transgene is not as well-characterized as that of the LacI. In mouse models carrying LacZ transgenes, the LacZ mutational reporter is recovered utilizing E. coli host cells that are sensitive to galactose and medium containing galactose28. A drawback of this system is that recovery of the LacZ target also involves restriction endonuclease digestion followed by ligation and electroporation of E. coli hosts, thereby making it difficult to adapt the system for small numbers of cells. Although it is not an absolute requirement for working with stem/progenitor cell populations (one can always start with more mice), if large numbers of cells are required (e.g. millions or more) it will quickly become impractical and cost-prohibitive. Also, the relatively large size of LacZ, while providing a sensitive mutational reporter, is cumbersome and more costly for DNA sequence analysis and determination of mutation spectra. A major advantage of this model however, is its ability to detect large deletions and insertions, as well as chromosomal rearrangements.
Since all cells in the LacI, &#934;X174 and LacZ transgenic mouse models carry the reporter system, any of these mouse models can be used to measure mutagenesis in any cell type of interest, including stem and progenitor cells, as long as they can be reliably harvested and in sufficient numbers. Because we had extensive experience with the LacI mouse model and the LacI mutation assay, we decided to pursue this system further for mutagenesis analysis in hematopoietic stem and progenitor populations.
The hematopoietic tissue is well-characterized in terms of cell surface phenotype of its individual components, including long-term repopulating stem cells, which are identifiable as the extremely rare population of Lin-IL7R-Sca-1+cKit++(LSK)/ Flk2-CD150+CD48- cells29. Mohrin et al.4 demonstrated that the slightly larger population of LSK/Flk2- cells are still good representatives for HSCs and significantly different from the most primitive committed myeloid progenitor (CMP) population when it comes to studying DNA repair. Moreover, when the HSC-enriched LSK (Flk-2+ and Flk-2-) cells were compared to the Lin-IL7R-Sca-1-cKit++ (LS-K progenitor cells), there was still a significant difference in NHEJ ability5 between the less pure, stem cell-enriched LSK population and the progenitor cells. In our study we use HSC-enriched LSK (Flk-2+ and Flk-2-) cells because we found that at least 2 x 105 cells are required for consistent, reliable results in this mutagenesis assay; this cell number is extremely difficult to obtain when one sorts the LSK/Flk2-CD150+CD48 population or even the LSK/Flk2- population (in terms of mice, costs and practicality). This protocol, based on the one originally developed by Kohler et al.18 describes in detail how the spontaneous DNA mutant frequency can be determined in LSK cells and defined populations of differentiated myeloid cells as well as unseparated bone marrow and spleen cells.

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			<td height="21" style="height:21px;width:252px;">LacI transgenic mice&#160;</td>
			<td style="width:245px;">BioReliance Corporation</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:252px;">RecoverEase DNA Isolation Kit, including the RNace-It ribonuclease cocktail</td>
			<td style="width:245px;">Agilent Technologies (Stratagene)</td>
			<td style="width:195px;">720202</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:252px;">Transpack Packaging Extract, including the orange and blue transpack tubes and the SCS-8 E. coli</td>
			<td style="width:245px;">Agilent Technologies (Stratagene)</td>
			<td style="width:195px;">200221</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA size standards &#8211; lambda ladder</td>
			<td>Bio Rad</td>
			<td>170-3635</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.025 mM pore size membranes&#160;</td>
			<td>Fisher (Millipore)</td>
			<td>VSWP 025 00</td>
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		<tr height="25">
			<td height="25" style="height:25px;">245 mm2 bioassay dishes (trays)&#160;</td>
			<td>Fisher (Corning)</td>
			<td>07-200-600</td>
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			<td height="21" style="height:21px;">NZY broth (powder)</td>
			<td>Fisher (Teknova)</td>
			<td>N1144</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Fisher</td>
			<td>BP1423-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Fisher</td>
			<td>E-3120-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N, N-dimethylformamide</td>
			<td>Fisher</td>
			<td>AC34843-5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal</td>
			<td>Research Products Intl Corp (RPI)</td>
			<td>B71800-10.0</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Roche</td>
			<td>3-115-852</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR Extender Taq Polimerase kit</td>
			<td>5 PRIME</td>
			<td>2200500</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Agencourt AMPure XP cleanup kit</td>
			<td>Beckman/coulter</td>
			<td>A63880</td>
		</tr>
	</tbody>
</table>

identifying dna mutations in purified hematopoietic stem/progenitor cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

The in vivo mutagenesis assay measures a rare event (mutant PFUs) among many events (all PFUs). By performing the assay with small number of cells, it is possible that the outcome is considerably influenced by false-positive and false-negative results. To address this issue we performed a serial dilution experiment with unfractionated bone marrow cells, harvested from three different animals. We measured the mutant frequency in the bone marrow of these animals using 1.4 x 106, 7.0 x 105, 3....

Watch this Scientific Journal Video about Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells at JoVE.com

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

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12.9K Views.  UT Health Science Center at San Antonio. Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model. The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants spontaneously arisen or after exposure to genotoxins.