How are bone marrow transplants transformed

Lentiviral CRISPR / Cas9-mediated genome editing for the study of hematopoietic cells in disease models

Summary

Protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR / Cas9 system for the rapid development of mouse model systems with hematopoietic system-specific gene modifications are described.

Abstract

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. With advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are specifically manipulated in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-extending line-negative bone marrow cells with a guide RNA (gRNA) that targets specific genes and a red fluorescent reporter gene (RFP), then these cells are transformed into lethally irradiated C57BL / 6 -Mice transplanted. Mice transplanted with lentivirus expressing non-targeted gRNA are used as controls. Transplantation of transduced hematopoietic stem cells is assessed by flow cytometric analysis of RFP positive peripheral blood leukocytes. With this method, 90% transduction of myeloid cells and 70% of lymph cells can be achieved after 4 weeks after transplantation. Genomic DNA is isolated from RFP positive blood cells, and portions of the target site DNA are amplified by PCR to validate genomic editing. This protocol offers a high throughput evaluation of hematopoiesis regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Introduction

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Many studies in hematology and immunology are based on the availability of genetically engineered mice, including conventional and conditional transgenic / knock-out mice that use hematopoietic systemic Cre drivers such as Mx1-Cre, Vav-Cre, and others. 1,2,3,4,5. These strategies require the establishment of new strains of mice, which can be time consuming and financially burdensome. While revolutionary advances in genome editing technology have enabled the creation of new mouse strains in just 3-4 months with the appropriate technical know-how6,7,8,9 Much more time is required to amplify the mouse colony before experiments are carried out. In addition, these procedures are costly. For example, Jackson Laboratory lists the current price for the production services for knock-out mice at $ 16,845 per strain as of December 2018. Therefore, methods that are more economical and efficient than traditional murine transgenic approaches are more advantageous.

The clustered, regularly interspacete short palindromic repeats / CRISPR-associated protein 9 technology (CRISPR / Cas9) has led to the development of new tools for fast and efficient RNA-based, sequence-specific genome editing. Originally discovered as a bacterial adaptive immune mechanism to destroy invading pathogen DNA, the CRISPR / Cas9 system was used as a tool to increase the effectiveness of genome editing in eukaryotic cells and animal models. A number of approaches have been used to deliver CRISPR / Cas9 machines into hematopoietic stem cells (i.e., electroporation, nucleofection, lipofection, viral delivery, and others).

Here, a lentivirus system is used to transduce cells as it is able to effectively infect Cas9-exeecting murine hematopoietic stem cells and package together the guide RNA expression construct, promoters, regulatory sequences and encoding genes. fluorescent reporter proteins (e.g. GFP, RFP). With this method, ex vivo gene editing of mouse hematopoietic stem cells was achieved, followed by successful bone marrow reconstitution in fatally irradiated mice10. The lentivirus vector used for this study expresses the Cas9 and GFP reporter genes from the common core EF1a promoter with an internal ribosomal entry point in front of the reporter gene. The Rna leader is expressed from a separate U6 promoter. This system is then used to identify insertion and deletion mutations in the candidate clone hematopoiesis driver genes Tet2 and Dnmt3a10to create. However, the transduction efficiency with this method is relatively low (approx. 5% -10%) due to the large size of the vector insert (13 Kbp), which reduces the transduction efficiency and the virus titers during production.

Other studies have shown that a larger viral RNA size negatively affects both virus production and transduction efficiency. For example, increasing the insert size by 1 kb is reported to decrease virus production by 50%, and transduction efficiency will decrease to greater than 50% for mouse hematopoietic stem cells11. Therefore it is advantageous to reduce the size of the viral insert as much as possible in order to improve the efficiency of the system.

This shortcoming can be overcome by using Cas9 transgenic mice, in which the Cas9 protein is either constitutively or inducibly expressed12. The constitutive CRISPR / Cas9 knock-in mice express Cas9 endonuclease and EGFP from the CAG promoter Rosa26 location in ubiquitous ways. Thus, a construct with sgRNA under the control of the U6 promoter and the RFP reporter gene under the control of the EF1a core promoter can be delivered with the lentivirus vector in order to achieve genome editing. With this system, the genes of hematopoietic stem cells were successfully processed, which shows a transduction efficiency of 90%. Thus, this protocol provides a quick and effective way to create mice in which targeted gene mutations are introduced into the hematopoietic system. While our lab mainly used this type of technology to investigate the role of clonal hematopoiesis in cardiovascular disease13,14,15to investigate, it is also malignant on studies hematologically16. In addition, this protocol can be extended to analyze how DNA mutations in HSPC affect other disease or developmental processes in the haematopoietic system.

To establish a robust lentivirus vector system, high tikern virus stocks and optimized conditions for the transduction and transplantation of hematopoietic cells are required. The protocol provides instructions for creating a high-titre virus stock in Section 1, for optimizing murine hematopoietic stem cell culture conditions in Section 2, bone marrow transplantation methods in Section 3, and for assessment of engraftment in Section 4.

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Protocol

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All animal-related procedures have been approved by the University of Virginia Institutional Animal Care and Use Committee (IACUC).

1. Generation and purification of lentivirus particles

NOTE: Lentivirus particles containing the optimized Guide RNA can be generated by Addgene's detailed protocols:

  1. Prepare a 1: 200 solution of collagen (0.0005%) in 1x PBS.
  2. Coat a 6-well plate with collagen solution and incubate at 37 ° C, 5%CO2 for 30 min.
  3. Seed 293T cells at a density of 1 x 106 Cells per well and incubate at 37 ° C, 5%CO2 for 2 h.
  4. To prepare the mixture of three transfection plasmids for a well, combine 0.9 g of lentivirus vector, 0.6 g of psPAX2 and 0.3 g of pMD2.G, and then achieve a total volume of 10 L by adding deionized water. Adjust the amounts according to the number of wells. The amount and ratio of each plasmid may need to be further optimized to meet the researcher's needs.
  5. Carefully add 50 l 1x PBS and 5 l of the diluted PEI MAX (1.0 mg / ml) to the plasmid mixture and incubate for 15 min at room temperature (RT) (Table 1).
  6. Add 1 ml DMEM to the mixture.
  7. Aspirate media from the 6-well plate, add 1 ml of plasmid mix and incubate at 37 ° C, 5%CO2 for 3 h.
  8. Replace the media with 2 ml of fresh DMEM and incubate at 37 ° C, 5%CO2 for 24 h.
  9. Add 1 ml of fresh DMEM and cook at 37 ° C, 5%CO2 Incubate for a further 24 h (total incubation time 48 h).
  10. Transfer the culture supernatant to a 50 ml tube and centrifuge at 3,000 x G for 15 min to remove free floating cells.
  11. Filter the supernatant through a 0.45 m filter.
  12. Transfer the filtrate to polypropylene centrifuge tubes.
  13. Ultracentrifuge at 4 ° C and 72,100 x G at rMax for 3 h.
  14. Carefully aspirate the supernatant and leave the white pellet behind.
  15. Expose the pellet to 100 L of serum-free hematopoietic cell expansion medium without aeration.
  16. Save a 10 L aliquot to measure the viral titer and store any remaining aliquots at -80 ° C until needed.
  17. Titrate the virus using a qPCR-based assay according to the manufacturer's instructions with the 10 L viral aliquot.

2. Isolation and transduction of line-negative cells from demouse bone marrow (Figure 1A)

NOTE: Typically, to isolate enough cells, pairs of the tibia, femur, and humeri will be harvested from each mouse. Pelvic and spinal bones can also be harvested as a source of line-negative cells.

  1. Isolation of bone marrow cells
    1. Euthanize 8-10 week old CRISPR / Cas9 male knock-in mice with 5% isoflurane followed by cervical dislocation, then disinfect their skin with 70% ethanol.
    2. Using dissecting scissors, make a cross section in the skin just below the rib cage and peel the skin in both directions to expose the legs and arms.
    3. Gently separate the lower limbs from the hipbone by twisting the hip joint. Cut along the head of the femur to completely remove the femur from the hip. Dislocate the knee and cut at the joint to separate the femur and tibia while leaving the osseous epiphysis intact. Dislocate the ankle and peel the foot and additional muscle.
    4. Cut over the shoulder with scissors to loosen the upper limbs. Dislocate the shoulder, then cut at the elbow joint to harvest the humeral bone.
    5. Use cellulosic fiber wipes to gently remove muscle from the thighbones, tibia, and humeri. Take extra precautions to make sure the bones don't break during this process.
    6. Place the isolated bones in a 50 ml conical tube with RPMI and put them on ice.
      NOTE: The following steps should be performed in a Biosafety Class II cabinet.
    7. Transfer the bones to a sterile, 100 mm culture dish.
    8. Grasp the bone with blunt forceps, and, using dissecting scissors, carefully cut both epiphyses.
      NOTE: Inadequate cutting will result in incomplete fusion of the bone marrow, while overly aggressive cutting will result in cell loss.
    9. Fill a 10 ml syringe with ice cold RPMI and use a 22 G needle to flush the bone marrow from the shaft into a new 100 mm culture dish.
      NOTE: Bones will turn white and translucent if the bone shaft has been flushed well. If not, cut the ends of the bones again and rinse them again.
    10. After the bone marrow is collected, make a unicellular suspension by passing the bone marrow through a 10 ml syringe with an 18 G needle several times. Repeat 10x to ensure a single cell suspension.
    11. Filter the cell suspension through a 70 m cell strainer into a 50 ml conical tube.
    12. Centrifuge at 310 x G for 10 min at 4 ° C.
    13. Aspirate the supernatant and place the cell pellets in a suitable volume of optimized separation buffer for the following cell separation process.
  2. Isolation and lentivirus transduction from line-negative cells
    NOTE: Mouse line-negative cells are derived from the bone marrow of Cas9 transgenic mice3 orisolated from other strains of mice using a lineage depletion kit following the manufacturer's instructions. Typically, line-negative cells make up 2% -5% of total bone marrow cells, and purity is usually greater than 90% after isolation. The isolated line-negative cells are cultured in serum-free hematopoietic cell expansion medium, supplemented with 20 ng / ml recombinant murine minimal tPO and 50 ng / ml recombinant murine SCF, then with the lentivirus vector for 16 h in the case of multiple infections (MOI) = 100 .
    1. To isolate line negative cells, use the feed cell depletion kit according to the manufacturer's instructions.
    2. After isolation, re-expose the line-negative cells to 1 ml of serum-free hematopoietic cell expansion medium.
    3. Seed the cells in a 6-well plate with a density of 1.5 x 106 Cells / ml (5 x 105 Lineage-negative cells / mouse).)
    4. Add recombinant murine TPO and SCF in wells at final concentrations of 20 ng / ml and 50 ng / ml, respectively.
    5. Pre-incubation cells at 37 ° C in 5%CO2 for 2 h.
    6. Add lentivirus at MOI = 100, 4 g / ml polybrene and penicillin / streptomycin to the wells and at 37 ° C, 5%CO2 for 16-20 h(Figure 1B)brood.
    7. The next day, collect the lentivirus-transduced cells into a 15 ml cone-shaped tube and centrifuge at 300 g for 10 min.
    8. Carefully aspirate the supernatant and replace the pellet in 200 L RPMI per mouse. Store cells at RT until transplanted into mice (Section 3).

3. Transplantation of transduced cells into lethally irradiated mice

  1. On the day of the bone marrow transplant, place the recipient mice in an eight-slice cake cage and expose them to two doses of total body radiation (550 rads / dose, total dose = 1100 rads), with approximately 4 hours between each radiation session.
  2. After the second irradiation session, project transduced line negative cells via the retroorbital venous plexus (200 L total) with an insulin syringe into each anesthetized recipient mouse (Figure 1C).
  3. After irradiation, mice should be housed in sterilized cages and given a soft diet and drinking water supplemented with antibiotics for 14 days.
  4. After 3-4 weeks after the bone marrow transplant, analyze peripheral blood to check for transplantation of transplanted donor cells (Section 4).

4. Evaluation of the peripheral blood chimera

  1. Anesthetize mice with 5% isoflurane and obtain a blood sample from a retro-orbital vein with capillary tubes and collect it in K.2EDTA tubes (the volume in a capillary tube is sufficient for the following test).
  2. Transfer 20 L blood from the K2EDTA tubes into the 5 ml round polystyrene test tubes and place on ice.
  3. Add 1.5 ml of RBC lysis buffer to lyse red blood cells. Incubate for 5 min on ice.
  4. To neutralize the lysis buffer, wash samples with FACS buffer (1.5 ml / sample).
  5. Centrifuge at 609 x G atr max for 5 min at 4 ° C. Discard the supernatant.
  6. Incubate the cells with a cocktail of monoclonal antibodies (diluted in 100 L FACS buffer / sample) at RT for 20 min in the dark. A full list of antibodies can be found in the section materials above.
  7. Wash the cells once with FACS buffer (2 ml / sample). Centrifuge at 609 x G atr max (1,800 rpm) for 5 min at 4 ° C. Discard the supernatant completely.
  8. Fix the cells with paraformaldehyde-containing fixation buffer (100 l / tube) for 10 min at 4 ° C.
  9. Wash cells once with FACS buffer (3 ml / sample). Centrifuge at 609 x G atr max (1,800 rpm) for 5 min at 4 ° C. Discard the supernatant completely.
  10. Suspend the pellet in 400 L FACS buffer.
  11. Keep the samples at 4 ° C until analyzed by flow cytometry.

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Representative Results

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Using the protocol described above, approximately 0.8-1.0 x 108 Bone marrow cells obtained per mouse. The number of line negative cells we get is roughly 3 x 106 Cells per mouse. Typically, the yield of bone marrow line negative cells is 4% -5% of total bone marrow nucleus cells.

The chimera of the transduced cells (RFP positive) is assessed by flow cytometry of the peripheral blood (Figure 2A,B.).Blood is isolated from the retroorbital vein and appropriate markers are used to determine the identity of each hematopoietic cell population (i.e., neutrophils, monocytes, T cells, etc.) (Figure 3A,B.). Genomic DNA can be isolated from RFP positive blood cells, and portions of the target site DNA can be amplified by PCR and subcloned into TA cloning vectors for sequence analysis. These plasmids are in E. coli transduced and the target site sequences are determined by Sanger sequencing (Figure 4). Alternatively, target location sequences can be determined by other methods, such as e.g. B. Sanger sequencing of the pooled genome, followed by tracking of Indels by decomposition analysis (TIDE)10. For the control state, mice are usually transplanted with cells that are transduced with a lentivirus that expresses non-targeted guide RNA.


illustration 1: Schematic representation of this protocol.(A) Isolation of line-negative bone marrow cells from Cas9-execting mice (Section 2.1). (B) Lentivirus transduction from line negative cells (Section 2.2). (C) Retroorbital injection of transduced cells into lethally irradiated wild-type mice (Section 3). Please click here to view a larger version of this image.


Figure 2:Efficient lentiviral transduction of mouse bone marrow line negative cells in vitro.(A) Flow cytometric analysis shows successful transduction of line-negative cells. The analysis was carried out after 7 days of in vitro culture. (B) On average, 75.7% of the cells were transduced in this test (n = 3). Please click here to view a larger version of this image.


Figure 3:Reconstitution of fatally irradiated mouse bone marrow by transduced line negative cells.(A) Flow cytometric analysis of mouse peripheral blood after reconstitution with hematopoietic stem cells transduced (bottom) or not (top) with lentivirus-excessive RFP. Neutrophils are defined as Ly6G+ and Ly6CHi monocytes as Ly6G- and Ly6C+, and B cells as CD45R+. (B) In these assays, an average of 94.8%, 93.5%, and 82.7% of the cells are RFP+ in the neutrophils, Ly6C Chi monocyte and B cell populations (n ​​= 8). Please click here to view a larger version of this figure.


Figure 4:ratinggene editing in transduced blood cells.(A) Example of gene editing showing sequencing results of mutated Dnmt3a locus in RPF positive blood cells. Deletions are referred to as red dashes and insertions with red letters. (B) Summary of the detected mutations. (C) 69% (11/16 clones) showed out-of-frame / premature stop mutations. Please click here to view a larger version of this image.

PlasmidSize (bp)Amount per well (g)relationship
pLKO5.077000.92
psPAX2106680.61
pMD2.G58220.31
PEI-max (stock: 100 mg / ml)5 l / well

Table 1: Amounts of plasmid and PEI-max used for transfection.

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Discussion

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The advantage of this protocol is the creation of animal models that harbor specific mutations in hematopoietic cells in a rapid and extremely inexpensive manner compared to traditional mouse transgenic approaches. It was found that this method enables the generation of mice with hematopoietic cell gene manipulations within one month. There are several important steps in this protocol that require further consideration.

Screening the gRNA sequence

It is recommended that gRNAs be tested in vitro to assess processing efficiency before performing in vivo experiments. The efficiency of gRNAs is tested with a cell-free in vitro transcription and screening system. The transcribed gRNA is validated by measuring its efficiency in gluing the template DNA in the presence of recombinant Cas9 protein using agarose gel electrophoresis. Commercially available kits are available for this purpose.

Indel mutations are characterized by the TA cloning of PCR products, which are amplified from the processed region, transform bacterial cells with these plasmids and pick up individual colonies for Sanger sequencing. However, this method is tedious and time consuming. Alternatively, next generation sequencing (NGS) or pooled DNA sequencing followed by TIDE analysis can be performed19. The TIDE algorithm was created to analyze Sanger sequence tracks generated from complex samples. It has been shown that Indel estimates with TIDE usually match those of NGS20are not targeted. The analysis software is online at

Generation of high-titer lentivirus particles

The viral vesicular stomatitis virus G-protein, which is essential for cell infection, is highly pH-sensitive. Therefore, it is important to keep the culture medium within an acceptable pH range and it should not develop a yellowish appearance. Collagen-coated dishes for virus generation are used because it speeds up the attachment of HEK293T cells and enables the transfection to be carried out in a few hours instead of waiting overnight. However, depending on the test plan, overnight incubation can also be considered.

Purification of lentivirus particles

In order to achieve efficient transduction of hematopoietic stem cells, it is necessary to generate high-titer lentivirus. Optimizing the centrifugation speed is a key feature. While the concentration of the lentivirus is usually 90,000 x Gseveral reports have shown that virus recovery increases when material is processed at the slower speed of 20,000x G18centrifuged. The production of high-titer lentivirus preparation without ultracentrifugation has also been suggested17. It should be noted that it is important to expose the virus centrifugation pellet while avoiding forceful pipetting to minimize aeration and to maintain virus integrity. High-titer lentivirus particles are required for efficient transduction of haematopoietic stem cells11. Pilot tests showed that an MOI of 100 is optimal in terms of reduction efficiency and cell viability. It is recommended that lentivirus stocks be assessed based on cell viability and transduction efficiency.

Storage of lentivirus particles

The lentivirus titer is highly temperature sensitive and the titer can be drastically reduced by inadequate storage conditions and repeated freeze-thaw cycles. The transduction efficiency of the lentivirus was found to decrease rapidly at 4 ° C [t (1/2) = 1.3 days] or more freeze-thaw cycles [t (1/2) = 1.1 rounds]. It is recommended to freeze the virus preparations in liquid nitrogen or crushed dry ice shortly after suspending the virus pellet. Virus stocks should be kept at -80 ° C and thawed on ice to RT just before equilibrium and11use.

There are several potential limitations to be aware of. First, the introduction of off-target indel mutations by CRISPR / Cas9 has long been appreciated. It has also been shown that CRISPR / Cas9 can cause off-target mutations in vivo21can induce. In practice, off-target indel mutations can be avoided by using gRNA sequences that closely match thought genome locations and have more than four incongruences to predicted secondary locations. Such a design can be done with existing silico tools22be performed. Other computational tools to predict gRNA with minimized off-target actions are available (

In addition to the traditional Indel mutations generated by CRISPR / Cas9, larger deletions in excess of kilobases have been reported. This can confuse studies; However, these larger deletions are reported to have much lower incidence compared to Indels23. Another potential problem is genetic compensation. It has been reported that mutated RNA with a premature termination codon (PTC) can upregulate related genes with sequence similarity by COMPASS complex-mediated activation of transcription24,25can lead. This event has been suggested as a mechanism that may lead to phenotypic differences between knock-out and knockdown approaches to gene ablation. Since CRISPR / Cas9-mediated genome editing relies heavily on the stochastic introduction of frame-shift mutations that lead to the generation of PTC, genetic compensation can alter the phenotype. To avoid genetic compensation, experiments can be considered that target the regulatory sequences of a gene from CRISPR / Cas9 or through the introduction of epigenetic modifiers with Cas9 as an RNA-guided DNA recognition platform.

Finally, it should be recognized that hematopoiesis from cells engraved in lethally irradiated mice may differ from the native conditions of hematopoiesis. In addition, irradiation can have systemic effects on the organism that can confuse the interpretation of experiments investigating the consequences of gene mutations in hematopoietic cells.

Researchers have used catalytically inactive Cas9 proteins (dCas9) as an "RNA-guided DNA recognition platform" and used dCas9 fusion proteins to localize effector domains to specific DNA sequences in order to either repress (CRISPRi) or activate (CRISPRa). Transcription of off-target genes26,27. While this protocol uses catalytically active Cas9 transgenic mice to introduce dsDNA cleavage into the genomic DNA sequence, the epigenetic modification can be used to repress or activate certain genes by adding dCas9 with chromatin modifier domains such as dCas9-KRAB or dCas9 -VP64. Alternatively, dCas9 can be used as a transcription repressor as its own by blocking transcription engines from accessing the gene site27. More recently, Zhou et al. transgenic DCas9-SunTag-p65-HSF1 (SPH) transgenic mice that express a modified version of an epigenetic activator fused with dCas9 and have shown that this CRISPRa system in vivo28is functional.

Our laboratory mainly uses this technology to investigate the role of clonal hematopoiesis in cardiovascular diseases. In proliferating tissue, somatic mutations in cancer driver genes can provide a cellular growth advantage and lead to abnormal clonal expansions. In the hematopoietic system, this process is referred to as "clonal hematopoiesis" and results in situations where a substantial portion of an individual's leukocytes are replaced by mutated clones. There is a growing appreciation that abnormal clonal expansions accelerate cardiovascular diseases such as atherosclerosis and heart failure and contribute to morbidity and all-cause mortality15,29.

Recently, a causal relationship between several of these somatic mutations and cardiovascular disease has been documented, and aspects of the underlying mechanisms have been identified10,13,14explained. However, these somatic mutations likely represent the "tip of the iceberg" as epidemiological studies have shown that many additional candidate genes are associated with clonal hematopoiesis and potentially increased cardiovascular disease mortality. Therefore, a systematic, higher throughput evaluation of the clonal hematopoiesis driver genes is required. Current studies on the causal relationship between clonal hematopoiesis and cardiovascular diseases are based on the analysis of mice with hematopoietic system-specific transgenic (Mx1-Cre, Vav-Cre, etc.) or mice after bone marrow transplantation. However, these strategies must establish new mouse colonies and can become a financial and physical burden on researchers. Therefore, a cheaper and faster method than the conventional murine transgenic / knock-out approach used in the past is justified. Lentiviral vectors used to transduce HSPC and CRISPR technologies to develop mutations, as described in this manuscript, facilitate the study of clonal hematopoiesis and cardiovascular disease.

In addition to the generation of conventional knock-out locus, this method can be applied to the production of cut mutant proteins. For example, researchers have successfully generated a hematopoietic Ppm1d cleavage, which is often seen in patients with clonal hematopoiesis, by frameshifting mutations with a gRNA targeting exon 6 of the Ppm1d gene30introduced.

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Disclosures

The authors have nothing to reveal.

Acknowledgments

S. S. was supported by an American Heart Association Postdoctoral Fellowship 17POST33670076. K. W. was supported by NIH Grants R01 HL138014, R01 HL141256, and R01 HL139819.

Materials

SurnameCompanyCatalog NumberComments
RPMI medium 1640 (1X)Gibco11875-093medium
Sulfamethoxazole and Trimethoprim injectionTEVA0703-9526-01
1/2 cc LO-DOSE INSULIN SYRINGEEXELINT26028general supply
293T cellsATCCCRL-3216--Cell line
APC-anti-mouse Ly6C (Clone AL-21)BD Biosciences560599Antibodies
APC-Cy7-anti-mouse CD45R (RA3-6B2)BD Biosciences552094Antibodies
BD Luer-Lok disposable syringes, 10 mlBD309604general supply
BD Microtainer blood collection tubes, K2EDTA addedBD Bioscience365974general supply
BD Precisionglide needle, 18 GBD305195general supply
BD Precisionglide needle, 22 GBD305155general supply
BV510-anti-mouse CD8a (Clone 53-6.7)Organic region100752Antibodies
BV711-anti-mouse CD3e (Clone 145-2C11)Organic region100349Antibodies
Collagen from calf skinSigma-Aldrich9007-34-5general supply
Corning Costar Ultra-Low Attachment Multiple Well Plate, 6 wellMillipore SigmaCLS3471general supply
CRISPR / Cas9 knock-in miceThe Jackson Laboratory028555mouse
DietGel 76AClear H2O70-01-5022general supply
Dulbecco’s Modified Eagle’s Medium (DMEM) - high glucoseSigma AldrichD6429medium
eBioscience 1X RBC Lysis BufferThermo fisher Scientific00-4333-57Solution
Falcon 100 mm TC-Treated Cell Culture DishLife sciences353003general supply
Falcon 5 mL round bottom polystyrene test tubeLife sciences352054general supply
Falcon 50 mL Conical Centrifuge TubesFisher Scientific352098general supply
Falcon 6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture PlateLife science353046general supply
Fisherbrand microhematocrit capillary tubesThermo Fisher Scientific22-362566general supply
Fisherbrand sterile cell strainers, 70 μmFisher Scientific22363548general supply
FITC-anti-mouse CD4 (Clone RM4-5)Invitrogen11-0042-85Antibodies
Fixation bufferBD Bioscience554655Solution
Guide-it Compete sgRNA Screening SystemsClontech632636Kit
Isothesia (Isoflurane) solutionHenry Schein29404Solution
Lenti-X qRT-PCR titration kitTakara631235Kit
Lineage Cell Depletion Kit, mouseMiltenyi Biotec130-090-858Kit
Millex-HV Syringe Filter Unit, 0.45 mmMillipore SigmaSLHV004SLgeneral supply
PBS pH7.4 (1X)Gibco10010023Solution
PE-Cy7-anti-mouse CD115 (Clone AFS98)eBioscience25-1152-82Antibodies
PEI MAXPolysciences24765-1Solution
Penicillin-Streptomycin MixtureLonza17-602FSolution
PerCP-Cy5.5-anti-mouse Ly6G (Clone 1A8)BD Biosciences560602Antibodies
pLKO5.sgRNA.EFS.tRFPAddgene57823Plasmid
pMG2DAddgene12259Plasmid
Polybrene Infection / Transfection ReagentSigma AldrichTR-1003-GSolution
Polypropylene Centrifuge TubesBECKMAN COULTER326823general supply
psPAX2Addgene12260Plasmid
RadDisk - Rodent Irradiator DiskBraintree ScientificIRD-P Mgeneral supply
Recombinant Murine SCFPeprotech250-03Solution
Recombinant Murine TPOPeprotech315-14Solution
StemSpan SFEMSTEMCELL Technologies09600Solution
TOPO TA cloning kit for sequencing with One Shot TOP10 Chemically Competent E.coliThermo fisher ScientificK457501Kit
Zombie Aqua Fixable Viability KitBioLegend423102Solution

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