Patent Application
20220386574
This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named 07039-2041001_ST25.txt. The ASCII text file, created on Jun. 3, 2022, is 26.7 kilobytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety.
This document relates to transgenic non-human animal models for cellular senescence, and to methods and materials for making and using the transgenic non-human animal models.
Cellular senescence is a cell fate characterized by essentially irreversible proliferative arrest (Gorgoulis et al., Cell 179:813-827, 2019). A variety of stimuli, including DNA damage, dysfunctional telomeres, oncogenic proteins, fatty acids, reactive oxygen species (ROS), mitogens, and cytokines, can act alone or in combination to drive cells into the cellular senescence fate through pathways involving p16/Rb (retinoblastoma), p53/p21, and possibly other factors (Tchkonia et al., J Clin Invest 123:966-972, 2013). These stimuli can contribute to widespread changes in gene expression that underlie senescence-associated growth arrest, the senescence-associated secretory phenotype (SASP), resistance to apoptosis, and changes in morphology (Tchkonia et al., supra; and Kirkland and Tchkonia, EBioMedicine 21:21-28, 2017). In these respects, cellular senescence can be considered a state of major cellular programming in addition to differentiation, proliferation, or apoptosis. Intracellular autocrine loops reinforce progression to irreversible replicative arrest, heterochromatin formation, and initiation of the pro-inflammatory SASP over a span of days to weeks (Tchkonia et al., supra; Kirkland and Tchkonia, supra; and Kuilman, et al., Cell 133:1019-1031, 2008).
Senescent cell burden increases in various tissues with aging (Wang et al., Aging Cell 8:311-323, 2009) and multiple chronic conditions (Zhu et al., Curr Opin Clin Nutr Met Care 17:324-328, 2014). Depending on specific tissues and varied pathological states, the percent of senescent cells can range from 1-20% (Baker et al., Nature 530:184-189, 2016; Xu et al., Elife 4:e12997, 2015; and Xu et al., Proc Natl Acad Sci USA 112:E6301-6310, 2015). Even when the percentage is relatively low, senescent cells can cause substantial tissue dysfunction (Kirkland et al., J Am Geriatr Soc 65:2297-2301, 2017). Senescent cells can elicit damage in both an autocrine and paracrine fashion, such that in addition to inducing intracellular dysfunction by autocrine signaling, senescent cells can be “contagious” and induce cellular senescence and the SASP in nearby non-senescent cells (Xu et al., Proc Natl Acad Sci USA 112:E6301-6310, 2015; Acosta et al., Nature Cell Biol 15:978-990, 2013; Nelson et al., Aging Cell 11:345-349, 2012; Xu et al., Nature Med 24:1246-1256, 2018; and da Silva et al., Aging Cell 18:e12848, 2019). Senescent cells also can directly impair the function of healthy stem cells (Xu et al., Elife 4:e12997, 2015). Paracrine signaling from senescent cells can amplify damage within tissues, possibly through the SASP, ROS, or other factors, which might partially explain why small number of senescent cells can be so harmful.
The INK-ATTAC (Baker et al., Nature 479:232-236, 2011) and p16-3MR (Demaria et al., Developmental Cell 31:722-733, 2014) transgenic mouse models have been used to investigate the role of senescent cells in vivo. Both models were designed using the p16 promoter to drive an inducible suicide gene, by which p16Ink4a-highly-expressing (p16high cells) can be eliminated in vivo. By leveraging these two models, the causal role of p16high cells has been suggested in a number of pathological conditions, including osteoporosis (Farr et al., Nature Med 23:1072-1079, 2017), metabolic dysfunction (Xu et al., Elife 4:e12997, 2015; and Palmer et al., Aging Cell e12950, 2019), osteoarthritis (Jeon et al., Nature Med 23:775-781, 2017), neurodegenerative diseases (Bussian et al., Nature 562:578-582, 2018), cardiac dysfunction (Baker et al. 2016, supra), kidney dysfunction (Baker et al. 2016, supra), vasomotor dysfunction (Roos et al., Aging Cell 15:973-977, 2016), atherosclerosis (Childs et al., Science 354:472-477, 2016), liver steatosis (Ogrodnik et al., Nature Commun 8:15691, 2017), pulmonary fibrosis (Schafer et al., Nature Commun 8:14532, 2017), stem cell dysfunction (Xu et al., Elife 4:e12997, 2015; and Chang et al., Nature Med 22:78-83, 2016), and lifespan reduction (Baker et al. 2016, supra). Two additional senescence-related transgenic mouse models have knock-in Cre inserted into the native p16 locus (Grosse et al., Cell Metab 32:87-99 e86, 2020; and Omori et al., Cell Metab 32:814-828 e816, 2020). Although valuable, these models only target p16high cells. However, not all p16high cells are senescent (Hall et al., Aging 9:1867-1884, 2017; and Frescas et al., Cell Cycle 16:1526-1533, 2017), and not all senescent cells express high levels of p16.
Disclosed herein is a p21-Cre mouse model that contains a p21 promoter driving an inducible Cre polypeptide. The mouse model can enable the examination of p21Cip1-highly-expressing (p21high) cells, a previously unexplored senescent cell population. p21high cells are distinct from p16high cells in a number of aged tissues, and exhibit several characteristics typical of senescent cells. By crossing p21-Cre mice with different floxed mice, p21high cells can be monitored, sorted, imaged, eliminated, or modulated in vivo. As demonstrated herein, for example, use of the p21-Cre mouse model showed that p21high cells can be induced by various conditions, and that percentages of p21high cells were higher (ranging from 1.5% to 10%) in a number of tissues in 23-month-old mice than the percentages (<1%) in 3-month-old mice. In addition, intermittent clearance of p21high cells improved physical function in 23-month-old mice. Thus, the studies described herein demonstrated that the p21-Cre mouse model is a useful tool for studying p21high cells to further understand the biology of senescent cells. Thus, this document provides methods and materials for modeling and studying cellular senescence.
For example, in some cases, this document provides transgenic non-human animals that can express a marker in senescent cells (e.g., p21high senescent cells). In some cases, this document provides transgenic non-human animals that can be induced to express a marker at a level that is directly proportional to the amount of senescent cells (e.g., p21high senescent cells) in the animals. In some cases, this document provides transgenic non-human animals that can be induced to delete senescent cells (e.g., p21high senescent cells). As described herein, transgenic mice can be produced to contain nucleic acid that allows for the controlled expression, detection, and/or clearance of senescent cells (e.g., p21high senescent cells) by, for example, controllably inducing apoptosis of senescent cells while inducing little, or no, apoptosis of non-senescent cells. For example, a transgenic non-human animal provided herein can be allowed to grow and develop for a period of time and then can be treated with a compound (e.g., tamoxifen) capable of inducing apoptosis of senescent cells within the transgenic animal while inducing little, or no, apoptosis of non-senescent cells within the transgenic animal. Clearance of senescent cells within a transgenic non-human animal can delay or reduce the likelihood of age-related disorders and can maximize healthy lifespan. In some cases, a transgenic non-human animal provided herein can include nucleic acid encoding a marker polypeptide (e.g., a fluorescent polypeptide such as a GFP) configured to be expressed by senescent cells with little, or no, expression by non-senescent cells.
In one aspect, this document features a transgenic non-human animal, the nucleated cells of which contain a transgene, where the transgene includes a p21 promoter sequence operably linked to a nucleic acid sequence encoding a Cre recombinase (Cre) polypeptide fused to a tamoxifen-inducible estrogen receptor (ERT2) domain. The transgene can further contain, 3′ of the nucleic acid sequence encoding the Cre polypeptide fused to an ERT2 domain, an internal ribosome entry site (IRES) and a nucleotide sequence encoding a marker. The marker can be a green fluorescent protein (GFP). The non-human animal can be a mouse. The p21 promoter sequence can have at least 95% sequence identity with SEQ ID NO:1. The transgene can be within an H11 genomic locus of the non-human animal. Visceral fat cells, brain cells, intestine cells, heart cells, liver cells, and/or skeletal muscle cells of the animal can express the Cre polypeptide fused to an ERT2 domain. Visceral fat cells, brain cells, intestine cells, heart cells, liver cells, and/or skeletal muscle cells of the animal can express the Cre polypeptide fused to an ERT2 domain and said marker.
In another aspect, this document features a nucleic acid containing a p21 promoter sequence operably linked to a nucleotide sequence encoding a Cre polypeptide fused to an ERT2 domain. The nucleic acid can further contain, 3′ of the nucleotide sequence encoding a Cre polypeptide fused to an ERT2 domain, an IRES and a nucleotide sequence encoding a marker. The marker can be a GFP. The p21 promoter sequence can have at least 95% sequence identity with SEQ ID NO:1. The Cre polypeptide can have an amino acid sequence at least 95% identical to SEQ ID NO:4. The ERT2 domain can have an amino acid sequence at least 95% identical to SEQ ID NO:6.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document relates to methods and materials involved in the evaluation and/or removal of senescent cells within a mammal. For example, this document provides transgenic non-human animals that can contain an exogenous nucleic acid that includes a p21 promoter sequence operably linked to a sequence encoding an inducible Cre polypeptide. Such non-human animals can be farm animals such as pigs, goats, sheep, cows, horses, and rabbits, rodents such as rats, guinea pigs, and mice, and non-human primates such as baboons, monkeys, and chimpanzees. The term “transgenic non-human animal” as used herein includes, without limitation, founder transgenic non-human animals as well as progeny of the founders, progeny of the progeny, and so forth, provided that the progeny retain the transgene. The nucleated cells of the transgenic non-human animals provided herein can contain a transgene that includes a p21 promoter sequence operably linked to a nucleic acid sequence encoding an inducible Cre polypeptide, where the Cre polypeptide is capable of causing recombination at loxP sites flanking a nucleotide sequence of interest at another genomic site within the animal. A P21 promoter sequence of a transgene described herein can drive Cre polypeptide expression in senescent cells while driving less, little, or no Cre polypeptide expression in non-senescent cells.
In some cases, the inducible Cre polypeptide capable of causing recombination at loxP sites can be a polypeptide that includes two polypeptide sequences fused together (e.g., a fusion polypeptide). For example, a fusion polypeptide can include a Cre polypeptide and an inducible estrogen receptor (ER) polypeptide (e.g., a tamoxifen-inducible ER polypeptide (ERT2), as described herein). In some cases, a transgene provided herein can further include nucleic acid encoding a marker polypeptide such as a fluorescent polypeptide (e.g., GFP, BFP, or RFP). For example, a transgene provided herein can include a p21 promoter operably linked to a sequence encoding a fusion polypeptide that includes Cre and an inducible ER polypeptide, followed by an internal ribosome entry site (IRES), followed by a sequence encoding a marker polypeptide (e.g., GFP).
The term “operably linked” as used herein refers to positioning a regulatory element (e.g., a promoter sequence, an inducible element, or an enhancer sequence) relative to a nucleic acid sequence encoding a polypeptide in such a way as to permit or facilitate expression of the encoded polypeptide. In the transgenes disclosed herein, for example, a promoter sequence (e.g., a p21 promoter sequence) can be positioned 5′ relative to a nucleic acid encoding a polypeptide (e.g., a Cre-ERT2 fusion polypeptide).
This document provides transgene nucleic acids, as well as transgenic non-human animals containing the transgenes, and methods for using the transgenic non-human animals. The transgene nucleic acids provided herein can contain a p21 promoter operably linked to a sequence encoding an inducible Cre polypeptide. In some cases, a transgene nucleic acid provided herein also can include an IRES and a nucleotide sequence encoding a marker.
The p21 promoter in the transgene constructs and transgenic non-human animals provided herein can have any appropriate length and any appropriate sequence, provided that it drives expression of an operably linked coding sequence under conditions that induce senescence. For example, a p21 promoter can be from about 3000 to about 3500 nucleotides in length (e.g., about 3000 to about 3100, about 3100 to about 3200, about 3200 to about 3300, about 3300 to about 3400, or about 3400 to about 3500 nucleotides in length). Further, the p21 promoter can have the sequence set forth in SEQ ID NO:1, or can have a sequence that is at least 95% identical (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identical) to the p21 promoter sequence set forth in SEQ ID NO:1:
The sequence encoding an inducible Cre polypeptide within the transgene constructs and transgenic non-human animals provided herein can have any appropriate length and any appropriate sequence, provided that after expression and induction, the Cre polypeptide can cause recombination at loxP sites within the cells in which the Cre polypeptide is expressed. In some cases, for example, an inducible Cre polypeptide can be a Cre-ERT2 fusion polypeptide. The Cre-ERT2 fusion polypeptide can have the amino acid sequence set forth in SEQ ID NO:2, or can have a sequence that is at least 95% identical (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO:2:
In some cases, the Cre-ERT2 polypeptide can be encoded by the nucleotide sequence set forth in SEQ ID NO:3, or can be encoded by a nucleotide sequence that is at least 95% identical (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identical) to the Cre-ERT2 nucleotide sequence set forth in SEQ ID NO:3:
In some cases, the Cre portion of the Cre-ERT2 fusion polypeptide can have the amino acid sequence set forth in SEQ ID NO:4, or can have a sequence that is at least 95% identical (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO:4:
The Cre portion of the Cre-ERT2 polypeptide can be encoded by the nucleotide sequence set forth in SEQ ID NO:5, or can be encoded by a nucleotide sequence that is at least 95% identical (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identical) to the nucleotide sequence set forth in SEQ ID NO:5:
In some cases, the ERT2 portion of the Cre-ERT2 fusion polypeptide can have the amino acid sequence set forth in SEQ ID NO:6, or can have a sequence that is at least 95% identical (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO:6:
In some cases, the ERT2 portion of the Cre-ERT2 polypeptide can be encoded by the nucleotide sequence set forth in SEQ ID NO:7, or can be encoded by a nucleotide sequence that is at least 95% identical (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identical) to the ERT2 nucleotide sequence set forth in SEQ ID NO:7:
When an IRES and a nucleotide sequence encoding a marker are included in the transgene constructs and transgenic non-human animals provided herein, any appropriate IRES sequence and marker coding sequence can be used. For example, a IRES can have the sequence set forth in SEQ ID NO:8, or a sequence that is at least 95% identical (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identical) to the IRES sequence set forth in SEQ ID NO: 8:
In some cases, the marker can be a fluorescent marker. For example, the marker can be a GFP, and can have the amino acid sequence set forth in SEQ ID NO:9, or can have an amino sequence that is at least 95% identical (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identical) to the sequence set forth in SEQ ID NO:9:
In some cases, the GFP can be encoded by the nucleotide sequence set forth in SEQ ID NO: 10, or by a nucleotide sequence that is at least 95% identical (e.g., at least 96%, at least 97%, at least 98%, or at least 99% identical) to the sequence set forth in SEQ ID NO: 10:
The percent sequence identity between a particular nucleic acid or amino acid sequence and a sequence referenced by a particular sequence identification number is determined as follows. First, a nucleic acid or amino acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained online at fr.com/blast or at ncbi.nlm.nih.gov. Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2. To compare two amino acid sequences, the options of Bl2seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence (e.g., SEQ ID NO:1), or by an articulated length (e.g., 20 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, an amino acid sequence that has 3100 matches when aligned with the sequence set forth in SEQ ID NO:1 is 96.2 percent identical to the sequence set forth in SEQ ID NO:1 (i.e., 3100÷3224×100=96.2). It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 is rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 is rounded up to 75.2. It also is noted that the length value will always be an integer.
Various techniques known in the art can be used to introduce transgenes into non-human animals to produce founder lines, in which the transgene is integrated into the genome. Such techniques include, without limitation, pronuclear microinjection (see, e.g., U.S. Pat. No. 4,873,191), retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci. USA 82:6148-1652, 1985), gene targeting into embryonic stem cells (Thompson et al., Cell 56:313-321, 1989), electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803-1814, 1983), and in vitro transformation of somatic cells, such as cumulus or mammary cells, followed by nuclear transplantation (Wilmut et al., Nature 385:810-813, 1997; and Wakayama et al., Nature 394:369-374, 1998). For example, fetal fibroblasts can be genetically modified to contain a p21-Cre construct (
Once transgenic non-human animals have been generated, expression of an encoded polypeptide (e.g., a Cre-ERT2 fusion protein and/or a marker polypeptide) can be assessed using any appropriate technique. For example, initial screening can be accomplished by Southern blot analysis to determine whether or not integration of the transgene has taken place. For a description of Southern analysis, see sections 9.37-9.52 of Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Press, Plainview; NY. Polymerase chain reaction (PCR) techniques also can be used for initial screening. PCR is described in, for example PCR Primer: A Laboratory Manual, ed. Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. Nucleic acids also can be amplified by ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplified. See, for example, Lewis, Genetic Engineering News 12:1, 1992; Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990; and Weiss, Science 254:1292-1293, 1991).
Expression of a nucleic acid sequence encoding a polypeptide (e.g., an inducible Cre polypeptide such as a Cre-ERT2 fusion polypeptide, or a marker polypeptide) in cells of transgenic non-human animals can be assessed using techniques that include, without limitation, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, Western blot analysis, immunoassays such as enzyme-linked immunosorbent assays, and reverse-transcriptase PCR (RT-PCR).
In some cases, as described herein, transgenic non-human animals containing a p21-Cre transgene can be crossed with other transgenic non-human animals containing a transgene that encodes a reporter (e.g., luciferase or tdTomato) or another polypeptide (e.g., a diphtheria toxin polypeptide, such as a diphtheria toxin A polypeptide), where the nucleotide sequence encoding the reporter or other polypeptide is separated from a promoter by a loxP-flanked STOP fragment. Progeny of the cross that are doubly transgenic can be selected and evaluated as described in the Examples herein. For example, expression and inducement of an inducible Cre polypeptide (e.g., a Cre-ERT2 fusion polypeptide) by cells within a doubly transgenic animal can lead to removal of the floxed STOP fragment and expression of the reporter or other polypeptide. It is understood that a particular phenotype in a transgenic or doubly transgenic animal typically is assessed by comparing the phenotype in the transgenic animal to the corresponding phenotype exhibited by a control non-human animal that lacks the transgene.
A transgenic non-human animal provided herein can have any appropriate genetic background.
This document also provides tissues (e.g., adipose, liver, intestine, brain, heart, and muscle) and cells (e.g., fat cells, preadipocytes, muscle cells, neuronal progenitor cells, hepatocytes, and endothelial cells) obtained from a transgenic non-human animal provided herein. Any appropriate method can be used to obtain senescent cells from a mammal. For example, senescent cells expressing a marker polypeptide (e.g., GFP) under the control of a p21 promoter sequence can be separated from non-senescent cells using techniques such as cell sorting methods based on the expression of the marker polypeptide. In some cases, cell lines of senescent cells can be used in place of freshly obtained senescent cells to identify agents having the ability to kill, or to facilitate the killing of, senescent cells and agents having the ability to delay, or reduce the likelihood of age-related disorders, and/or maximize healthy lifespan as described herein. In some cases, senescent cells can be obtained by cell passage in culture (e.g., greater than about 12, greater than about 15, or greater than about 20 cell passages). In some cases, senescent cells can be obtained from p21-Cre transgenic non-human animals fed a HFD or treated with DOXO. In some cases, senescent cells can be obtained from older p21-Cre transgenic non-human animals (e.g., p21-Cre mice that are at least 18, at least 20, or at least 23 months of age).
In some cases, senescent cells can be exposed to one or more test agents to identify agents having the ability to kill, or to facilitate the killing of, the senescent cells. Once identified as having the ability to kill, or to facilitate the killing of, the senescent cells, the identified agent can be applied to comparable non-senescent cells in comparable concentrations to confirm that the agent has a reduced ability to kill, or to facilitate the killing of, non-senescent cells. Those agents having the ability to kill, or to facilitate the killing of, senescent cells, with a reduced or no ability to kill, or to facilitate the killing of, non-senescent cells, can be classified as being agents having the ability to delay, or reduce the likelihood of age-related disorders, and/or maximize healthy lifespan. In some cases, senescent cells obtained from a transgenic mammal provided herein and treated in a manner that results in senescent cell death can be used as positive controls.
This document also provides methods and materials for identifying molecules (e.g., polypeptides, carbohydrates, lipids, and nucleic acids) possessed or expressed by senescent cells. For example, senescent cells can be obtained as described herein and assessed to identify molecules (e.g., polypeptides) possessed or expressed by those senescent cells. Any appropriate method can be used to identify molecules possessed or expressed by senescent cells. In some cases, for example, RNA-seq analysis can be used to identify molecules expressed by senescent cells. In some cases, polypeptide isolation and sequencing techniques can be used to identify polypeptides expressed by senescent cells.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
p21-Cre mouse model generation: A 7 kb DNA fragment, containing a 3225 bp mouse p21 promoter fragment followed by a nucleotide sequence encoding Cre fused to a tamoxifen-inducible estrogen receptor (ERT2) domain, was synthesized by GenScript (Piscataway, N.J.). An IRES followed by an open reading frame (ORF) coding for enhanced green fluorescent protein (EGFP) also was added into the transgene. The synthesized fragment (p21-ER-Cre) was subcloned into vector pBT378 for generating p21-Cre mice through Integrase-mediated transgenesis (IMT). Briefly, the p21-ER-Cre-pBT378 construct was microinjected into the pronucleus of recipient zygotes containing attP sites in the Hipp11 locus, which has a high recombination rate and results in stable expression of a single copy of the transgene. Site-specific recombination occurred between the attB sites in the plasmid and attP sites in the H11 genomic locus. Positive embryonic stem (ES) cell clones were then implanted into C57BL/6 females. A positive founder mouse was confirmed by PCR using a set of primers flanking the recombined 5′- and 3′-attP/attB sites in the H11 locus, as well as sets of specific primers that amplify unique sequences within the transgene.
Mouse models and drug treatments: All mice were maintained in a pathogen-free facility at 23-24° C. under a 12-hour light, 12-hour dark regimen with free access to a RCD (Teklad global 18% protein, Envigo #2918, Indianapolis, Ind.), or a 60% (by calories) HFD (D12492, irradiated; Research Diets, New Brunswick, N.J.) and water. For tamoxifen treatment, tamoxifen (Sigma-Aldrich, St Louis, Mo.) was dissolved in corn oil, and was administrated to mice by intraperitoneal (i.p.) injection once daily (2 mg per mouse) for two consecutive days. For DOXO treatment, DOXO (Sigma-Aldrich) was given to mice (20 mg/kg) once by i.p. injection. Tamoxifen was given in one dose at the same time of DOXO administration, and one more dose was given 16 hours after DOXO administration. Floxed tdTomato mice (#007914), floxed LUC mice (#005125), floxed DTA mice (#009669), CAG-Cre mice (#004682), and Relafl/fl mice (#024342) were purchased from the Jackson Laboratory (Bar Harbor, Me.).
Single cell RNA-seq analysis: Single cell RNA-seq data was collected from the Tabula Muris Senis single cell transcriptomic atlas for aged mice (Tabula Muris, Nature 583:590-595, 2020). Data were visualized using the provided browser based platform available at tabula-muris-senis.ds.czbiohub.org. In brief, single cell RNA seq data were examined from visceral adipose tissue (fluorescence-assisted cell sorting (FACS) method), liver (droplet method), and heart (droplet method). To account for differences in animal ages available for each tissue, all available data from animals 18 months or older was included in the analysis (Fat: 18 and 24 months; Liver: 18, 21, 24, 30 months; Heart: 18, 21, 24, 30 months). Expression profiles were then captured for p21 and p16 in each tissue.
Tissue dissociation: Visceral fat and liver tissues were minced with scissors, and digested in PBS containing 1 mg/ml type II collagenase (Sigma-Aldrich) and 10 μg/ml DNase I (Sigma-Aldrich) at 37° C. for 1 hour. After digestion, SVF cells were separated from visceral fat. SVF cells and liver cells were washed with phosphate buffered saline (PBS)/2% fetal bovine serum (FBS) and filtered through a 100 μm cell strainer. Cells were then incubated with ammonium-chloride-potassium (ACK) lysing buffer (Thermo Fisher Scientific, Waltham, Mass.) at room temperature for 10 minutes to remove red blood cells. Cells were washed with PBS/2% FBS for further experiments.
SA-β galactosidase staining. SA-β gal staining was assayed as described elsewhere (Xu et al., Proc Natl Acad Sci USA 112:E6301-6310, 2015; and Biran et al., Aging Cell 16:661-671, 2017). Briefly, purified SVF cells were fixed in 4% paraformaldehyde (PFA, Thermo Fisher Scientific) for 15 minutes at room temperature. Cells were washed with PBS, then incubated with SA-β gal staining solution containing 1 mg/ml X-gal, 40 mM citric acid/sodium phosphate at pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2 at 37° C. in a humidified chamber, and protected from light. After 16 hours of incubation, cells were washed in ice-cold PBS to stop the enzymatic reaction and resuspended in PBS buffer for ImageStreamX analysis.
EdU staining. Mice fed HFD for 5 months were treated with 1 mg/kg 5-ethynyl-2′-deoxyuridine (EdU, Cayman Chemical, Ann Arbor, Mich.) via intraperitoneal injection. After 20 hours, SVF cells were isolated and fixed in 4% PFA for 15 minutes at room temperature. After being washed with PBS/1% BSA, cells were permeabilized by 0.3% Triton X-100, and stained with 100 mM Tris, 2 mM CuSO4 (Sigma-Aldrich), 10 mM ascorbic acid, 2 μM Alexa Fluor 647 azide for 30 minutes at room temperature. Cells were washed with PBS/1% BSA twice, incubated with 0.1 μg/ml DAPI (Sigma-Aldrich) for 5 minutes, and analyzed by ImageStreamX.
Imaging flow cytometry analysis: SVF cells stained with SA-β gal and EdU were imaged by ImageStreamX Mark II (Amnis, Seattle, Wash.) and analyzed by IDEAS 6.2 software (Amnis). To focus cells, samples were gated using gradient RMS values of brightfield channel. Cells were further gated using brightfield area and aspect ratio to select single cells. GFP− and GFP+ SVF cells were measured for cell areas using brightfield area. Mean pixel of brightfield was used to calculate SA-β gal intensity in GFP− and GFP+ SVF cells as described elsewhere (Biran et al., supra). To measure EdU, the intensity of APC channel was compared between GFP− and GFP+ SVF cells.
Cell culture: Ear fibroblasts were isolated as described elsewhere (Xu et al., J Gerontol A Biol Sci Med Sci 72:780-785, 2017). To induce senescence, ear fibroblasts were treated with 0.5 μM DOXO for 24 hours, and considered to be senescent 10 days after.
Immunofluorescence staining: SVF cells were fixed in 4% PFA for 15 minutes at room temperature. Cells were then washed with PBS prior to a 10 minute incubation in permeabilization buffer (0.2% Triton X-100, 1% BSA in PBS). Next, the cells were washed with PBS and blocked for 1 hour in PBS/1% BSA solution. After blocking, cells were incubated with anti-Lamin B1 primary antibody (Proteintech, Rosemont, Ill.) 1:100 dilution in PBS/1% BSA overnight at 4° C. The next day, cells were washed with PBS and incubated for 1 hour at room temperature with Alexa Fluor 647-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific) diluted 1:200 in PBS/1% BSA. Stained cells were then washed with PBS and analyzed by flow cytometry.
Flow cytometry analysis: SVF cells and liver cells were isolated from visceral fat and liver tissues, and washed with PBS/2% FBS. Cells were stained with 0.1 μg/ml of DAPI for 5 minutes and detected by BD LSR II flow cytometer (BD Biosciences, San Jose, Calif.). Data analysis was performed using FlowJo V10.7 software.
FACS: SVF cells were isolated from mice fed HFD for 5 months, and were sorted into GFP− and GFP+ populations through BD FACSAria II flow cytometer (BD Biosciences). Sorted cells were used to detect p21, p16 and SASP expression.
Bioluminescence imaging: Mice were anesthetized with isoflurane gas (Piramal Critical Care, Bethlehem, Pa.) and injected intraperitoneally with 3 mg D-luciferin (Gold Biotechnology, St. Louis, Mo.) in 200 μl PBS. Five (5) minutes after injection, mice were placed in an IVIS spectrum in vivo imaging system (PerkinElmer, Waltham, Mass.) and bioluminescence images were subsequently captured with a 3 minute exposure time. The region of interest (ROI) was manually selected in a consistent way for individual mice in the same cohort. Bioluminescent signal of ROI was calculated using the Living Image 4.5.5 software (PerkinElmer).
Histological analysis: Visceral fat, liver, intestine, brain, heart, and muscle tissues were fixed with 4% PFA overnight at 4° C. After being washed with PBS, tissues were transferred to 30% sucrose (Sigma-Aldrich), incubated overnight at 4° C., and embedded in OCT compound (Thermo Fisher Scientific). Liver, intestine, brain, heart, muscle tissues in OCT blocks were cut into 6 μm thickness sections on a Leica CM3050 S cryostat, and visceral fat tissues were cut into 10 μm thick sections. Nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific) and imaged on a fluorescence microscope (Zeiss, Jena, Germany). To quantify tdTomato+ cells, three sections from each mouse were scanned and then counted using ImageJ software.
Physical function measurements: Physical function measurements were performed as described elsewhere (Xu et al., Nature Med 24:1246-1256, 2018). To test muscle strength and neuromuscular function, maximal walking speed, grip strength and grid hanging test were performed in aged mice. Maximal walking speed was tested with an accelerating 4 lane RotaRod system (Columbus Instruments, Columbus, Ohio). Mice were trained on the RotaRod for 2 consecutive days at a constant speed of 4 r.p.m. for 300 seconds. The day after the last day of training, mice were acclimatized to the testing room for 30 minutes. RotaRod test was started at 4 r.p.m. and accelerated from 4 to 47.2 r.p.m. in 6 minutes. The speed was recorded when the mouse fell off, and the average of 4 trials were calculated and normalized to the baseline speed. Forelimb grip strength (g) was assessed by a grip strength test meter (Bioseb, France). Results were averaged over 10 trials. A grid hanging test was performed on a grid placed onto a holding apparatus at a 35 cm distance from the floor, using a soft pad to avoid injuries. Hanging time was recorded when the mouse fell off, and the average of 3 trials were calculated and normalized to body weight as hanging duration (seconds)×body weight (g). A threshold at 7 minutes was set to consider the maximum hanging time.
Comprehensive laboratory animal monitoring system (CLAMS): Daily activity, food and water intake, metabolic performance, temperature were measured for the 23 month-old PL and PLD mice on a CLAMS system equipped with an Oxymax open-circuit calorimeter (Columbus Instruments, Columbus, Ohio). Physiological and behavioral parameters for individual mice were monitored over a 24 hour period (12 hour light/dark cycle), and results were analyzed by CLAX software (Columbus Instruments).
DNA preparation and PCR: Rela and P-Rela mice were sacrificed 24 hours after tamoxifen injection for 3 doses. Genomic DNA was extracted by lysing SVF cells in an alkaline reagent containing 25 mM NaOH and 0.2 mM EDTA at 94° C. for 2 hours and neutralizing with an equal volume of 40 mM Tris-HCl (pH 7.4), followed by purifying with isopropanol and 75% ice-cold ethanol (Sigma-Aldrich). An equal amount of DNA for each sample was adopted to react with GoTaq Green Master Mix (Promega, Madison, Wis.) and the indicated genotyping primers, respectively. PCR products were separated on a 2% agarose gel with SYBR safe DNA gel stain (Thermo Fisher Scientific) running in TAE buffer (Bio-Rad, Hercules, Calif.) and visualized using the ChemiDoc MP imaging system (Bio-Rad). All primers were purchased from Integrated DNA Technologies (IDT, Coralville, Iowa). Primer sequences are shown in TABLE 1.
RNA extraction and real-time PCR: Cells were collected, lysed in TRIzol reagent (Thermo Fisher Scientific), and extracted with chloroform, isopropanol, and 75% ice cold ethanol (Sigma-Aldrich). RNA was dissolved in RNase free water and reverse transcribed to cDNA with M-MLV reverse transcriptase kit (Thermo Fisher Scientific). Quantitative real-time PCR was conducted in four or five replicates using PerfeCTa FastMix II (Quantabio, Beverly, Mass.) on the CFX96 Real-Time PCR detection system (Bio-Rad). The relative mRNA level of target genes was normalized to TATA-binding protein (Tbp) and calculated via the 2−ΔΔCT method. Probes and primers for Tbp, p21, p16,116, Cxcl1, Ccl2 and Rela were purchased from Integrated DNA Technologies (IDT, Coralville, Iowa):
Senescent cells are highly heterogeneous in their biological properties, tissue distribution and responses to varied therapies (Roy et al., Cell 183:1143-1146, 2020). To explore the possible overlap of p16high cells and p21high cells or their respective population diversity, a single cell transcriptomic (SCT) atlas database (Tabula Muris Senis; Tabula Muris, Nature 583:590-595, 2020) was used. This database includes transcriptomic data from a range of tissues in 18-30 month-old mice. p21 and p16 expression levels were visualized using the browser-based interactive platform (tabula-muris-senis.ds.czbiohub.org/). In aged visceral fat, p21high cells are mainly endothelial cells, mesenchymal stem cells, and myeloid cells, while p16high cells are scarce. In liver, p21high cells are mainly Kupffer cells and myeloid cells, while p16high cells are mainly natural killer (NK) cells and a different population of Kupffer cells. In heart, p21high cells are mainly endothelial cells, while p16high cells are mainly leukocytes (
To generate the p21-Cre mouse model, a 7 kb DNA fragment (
An advantage of this site-specific transgenic approach using the H11 locus is that it was less likely to interfere with or disrupt any endogenous gene, as opposed to the use of random insertion or insertion into targeting gene locus. In addition, this approach allowed for the design of genotyping primers to distinguish +/+, p21-Cre/+, and p21-Cre/p21-Cre genotypes (
To validate function of the transgene, the p21-Cre mouse was crossed with floxed knock-in firefly luciferase (LUC) mice (Safran et al., Mol Imaging 2:297-302, 2003). Floxed knock-in LUC mice contain a loxP-flanked STOP fragment between the Gt(ROSA)26Sor (ROSA) promoter and LUC, which prevents LUC expression without the presence of Cre (
Metabolic stress and obesity can induce cellular senescence (Xu et al., Nature Med 24:1246-1256, 2018) and p21 expression (Schafer et al., Diabetes 65:1606-1615, 2016). Studies were conducted to examine whether p21high cells could be detected in PL mice under metabolic stress. PL mice were fed HFD for 4 months and then treated with two doses of tamoxifen. HFD led to significantly increased BLI signals in PL mice when compared to normal chow diet (NCD), indicating accumulation of p21high cells with obesity (
To image p21high cells using fluorescence in vivo at the tissue level, p21-Cre mice were crossed with floxed knock-in tdTomato mice (Madisen et al., Nat Neurosci 13:133-140, 2010), which contain a loxP-flanked STOP fragment between the CMV early enhancer/chicken p actin (CAG) promoter and tdTomato, yielding p21-Cre/+; tdTomato/+ (PT) mice (
p21high cells can also be detected by flow cytometry. Consistent with the fluorescence imaging, flow cytometry analysis revealed that the percentage of tdTomato+ p21high cells in visceral fat and liver was higher in old mice than in young PT mice (
p21high adipose-derived mesenchymal stem cells (ADSCs) have been shown to accumulate in aged mice (Wang et al., Aging Cell e13106, 2020). Single cell transcriptomics revealed that these naturally occurring p21high cells appear to exhibit altered pathways commonly observed in senescent cells, including Senescent Cell Anti-Apoptotic Pathways (SCAPs; increased cell survival and decreased apoptosis) and NF-κB, IL6/JAK, mTOR, FOXO, and HMGB1 pathways. Studies were conducted to further characterize p21high cells using the p21-Cre mouse model in vivo. PL mice were fed a HFD for 5 months to induce p21high cells in visceral fat (
To enable elimination of p21high cells in a temporal manner in vivo, PL mice were crossed with floxed diphtheria toxin A (DTA) mice (Voehringer et al., J Immunol 180:4742-4753, 2008), which contain a floxed-STOP cassette followed by DTA driven by the ROSA promoter. p21-Cre/+; LUC/DTA (PLD) mice were generated (
The role of senescent cells has been extensively examined across a range of pathological conditions, but the underlying mechanisms have rarely been investigated in vivo. The SASP is thought to be one of the major mechanisms responsible for the harmful effects of senescent cells (Tchkonia et al., J Clin Invest 123:966-972, 2013). Studies were conducted to determine the effects of the p21-Cre mouse model provided herein on the SASP exclusively in p21high cells. The NF-κB pathway serves as a master regulator of the SASP (Chien et al., Genes Dev 25:2125-2136, 2011), and Rela (v-rel reticuloendotheliosis viral oncogene homolog A, or p65) is a crucial subunit for NF-κB activation (Chen and Greene, Nat Rev Mol Cell Biol 5:392-401, 2004). Both Rela and the NF-κB pathway are highly activated in p21high preadipocytes isolated from aged mice (Wang et al., Aging Cell e13106, 2020). To inactivate the NF-κB pathway in p21high cells, p21-Cre mice were crossed with floxed Rela mice (Heise et al., J Exp Med 211:2103-2118, 2014), in which exon 1 of the Rela gene is flanked by loxP sites (
Next, studies were conducted to determine whether the Rela mutation led to SASP inhibition. CAG-Cre mice (Hayashi and McMahon, Devel Biol 244:305-318, 2002), which carry a constitutively active CAG promoter driving Cre, were crossed with floxed Rela mice to generate CAG-Cre/+; Relafl/fl (CAG-Rela) mice. Ear fibroblasts were isolated from these mice and senescence was induced using DOXO. After 4-OH treatment to induce Cre, expression levels of Rela and several key SASP components were observed to be reduced by 40-70% in senescent CAG-Rela cells compared to senescent WT cells (
Physical function typically declines with aging, leading to physical frailty, compromised systemic homeostasis, and increased vulnerability to stresses (Fried et al., Nature Aging 1:36-46, 2021). To investigate whether p21high cells play a causal role in physical frailty with aging, 20-month-old PL and PLD mice were treated with two doses of tamoxifen per month for three months (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Patent Application Ser. No. 63/196,390, filed on Jun. 3, 2021. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
| Number | Date | Country | |
|---|---|---|---|
| 63196390 | Jun 2021 | US |
