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. Author manuscript; available in PMC: 2020 Mar 1.
Published in final edited form as: Genesis. 2019 Jan 23;57(3):e23281. doi: 10.1002/dvg.23281

Using CRISPR/Cas9 engineering to generate a mouse with a conditional knockout allele for the promyelocytic leukemia zinc finger transcription factor

Lan Hai 1,#, Maria M Szwarc 1,#, Denise G Lanza 2, Jason D Heaney 2, John P Lydon 1,*
PMCID: PMC6422732  NIHMSID: NIHMS1013296  PMID: 30628160

Abstract

The promyelocytic leukemia zinc finger (PLZF) transcription factor mediates a wide-range of biological processes. Accordingly, perturbation of PLZF function results in a myriad of physiologic defects, the most conspicuous of which is abnormal skeletal patterning. Although whole body knockout of Plzf in the mouse (PlzfKO) has significantly expanded our understanding of Plzf function in vivo, a conditional knockout mouse model that enables tissue or cell-type specific ablation of Plzf has not been developed. Therefore, we used CRISPR/Cas 9 gene editing to generate a mouse model in which exon 2 of the murine Plzf gene is specifically flanked (or floxed) by LoxP sites (Plzff/f). Crossing our Plzff/f mouse with a global cre-driver mouse to generate the Plzfd/d bigenic mouse, we demonstrate that exon 2 of the Plzf gene is ablated in the Plzfd/d bigenic. Similar to the previously reported PlzfKO mouse, the Plzfd/d mouse exhibits a severe defect in skeletal patterning of the hindlimb, indicating that the Plzff/f mouse functions as designed. Therefore, studies in this short technical report demonstrate that the Plzff/f mouse will be useful to investigators who wish to explore the role of the Plzf transcription factor in a specific tissue or cell-type.

Keywords: promyelocytic leukemia zinc finger, mouse, CRISPR/Cas9, homology directed repair, conditional allele, cre recombinase, skeletal patterning, hindlimb, polydactyly

1. INTRODUCTION

Promyelocytic leukemia zinc finger (PLZF; also known as ZBTB16 or ZNF145) is a member of the evolutionary conserved POK (POZ and Kruppel) family of pleiotropic C2H2-type zinc finger transcription factors; reviewed in (Liu et al., 2016; Suliman et al., 2012). At the N-terminus, the BTB/POZ domain is responsible for homo- and hetero-dimerization, chromatin remodeling and epigenetic transcriptional control, formation of high-molecular weight DNA-protein complexes, and nuclear sub-localization. Positioned within the C-terminal region of PLZF, the nine C2H2 zinc finger motifs facilitate direct sequence-specific DNA binding to target genes. Cell and signaling context dependent, PLZF can serve as a transcriptional activator or repressor (Fahnenstich et al., 2003; Gaber et al., 2013; Ikeda et al., 2005; Kommagani et al., 2016; Labbaye et al., 2002; Liu et al., 2016; Mao et al., 2016; Singh et al., 2015; Suliman et al., 2012; Szwarc et al., 2018; Wang et al., 2012). Given its pleiotropic effects, PLZF drives a broad spectrum of developmental processes and physiological responses, including limb skeletal patterning, innate immune cell development, hematopoiesis, and spermatogenesis; reviewed in (Suliman et al., 2012). Underpinning these physiologies, PLZF modulates stem cell self-renewal, cell-cell communication, proliferation, differentiation, and apoptosis; reviewed in (Suliman et al., 2012).

The most conspicuous phenotype displayed by the Plzf knockout (PlzfKO) mouse is a severe defect in normal skeletal patterning of the hindlimb (Barna et al., 2000; Barna et al., 2005). This skeletal defect is also reported for the Luxoid (lu/lu) mouse (Green, 1955), which carries a naturally occurring nonsense mutation in the Plzf allele as well as for the rat with a naturally occurring cis-regulatory Plzf mutation ((lx/lx) (Kren, 1975; Liska et al., 2016)). The PlzfKO mouse exhibits homeotic-like anterior-to-posterior transformations in the axial and appendicular skeleton, causing hindlimb abnormalities such as fused or extra digits, loss of the tibia or conversion of the fibula to a tibia-like structure, and kinked tails (Barna et al., 2000). Subsequent analysis also revealed that these mutant mice exhibit impairments in the development of hematopoietic stem cell and neural progenitors (Gaber et al., 2013; Vincent-Fabert et al., 2016), in spermatogonial self-renewal (Buaas et al., 2004; Costoya et al., 2004), and in development of innate-like features of invariant natural killer T (iNKT) cells (Kovalovsky et al., 2008; Savage et al., 2011; Savage et al., 2008; Xu et al., 2009). Important from a translational perspective, many of these phenotypes also arise in humans with loss-of-function PLZF mutations (Fischer et al., 2008), highlighting the evolutionary conserved role of PLZF.

Despite tremendous advances in our understanding of Plzf function using the PlzfKO mouse, a conditional knockout mouse model that enables tissue or cell-type specific ablation of Plzf has yet to be developed. To address this issue, we describe in this short technical report the generation—through the use of Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas)9 genome editing—of a Plzf floxed (Plzff/f) mouse that is engineered to facilitate cre-mediated Plzf abrogation in a tissue or cell-type specific manner.

2. RESULTS AND DISCUSSION

We previously created conditional null alleles using a traditional homologous recombination strategy in mouse embryonic stem cells (Fernandez-Valdivia et al., 2010; Hashimoto-Partyka et al., 2006). However, due its speed, simplicity, precision, and significantly lower cost, the CRISPR/Cas9 genome engineering approach was used to generate a conditional Plzf knockout allele in the mouse. The strategy to create the conditional Plzf knockout allele was to flox exon 2 of the murine Plzf gene since exon 2 encodes greater than 50% of the protein, which includes the initiating ATG, the N-terminal BTB/POZ domain, and the first two C2H2 zinc fingers of the DNA binding domain (Barna et al., 2000). Exon 2 was also chosen because knockout of this exon using conventional embryonic stem cell gene targeting methods results in a null Plzf allele (Barna et al., 2000). To insert LoxP sites to flank exon 2 of the mouse Plzf gene, Cas9 mRNA, two separate and validated single guide RNAs (sgRNAs) targeted to intronic sequences flanking exon 2, two single-stranded oligonucleotide (ssODN) donors, each containing the 34 base-pair LoxP consensus site flanked asymmetrically by homology arms to each of the desired insertion sites (Figure 1 (Richardson et al., 2016)) were co-injected into approximately 200 single-cell C57BL/6NJ pronuclear-stage zygotes before their transfer into ICR recipient pseudopregnant females. To minimize the probability of off-target events, only sgRNAs predicted to have off-target sites with three mismatches or more were used to target Cas9 endonuclease activity to intronic sequences flanking exon 2 (Figure 2a); the location of the 5’ and 3’ sgRNAs in relation to exon 2 of the mouse Plzf gene is shown in Support Information Figure S1.

FIGURE 1.

FIGURE 1.

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Design and location of the 5’ and 3’ ssODNs used to generate the mouse Plzf conditional knockout allele. (a) Sequence of the 5’ target site in intron 1 of the mouse Plzf allele. Sequences highlighted in gray (36 base pairs (bp)) and blue (91bp) represent the asymmetric homology arms that are distal and proximal relative to the location of the 3’ protospacer adjacent motif (PAM). The ssODN is complementary to the non-target strand. The sequence of the 5’ target site is highlighted in green and underlined; the 3’ PAM (TGG) site is in red. Box includes the sequence (in the 5’ to 3’ direction) of the ssODNs for CRISPR/Cas9 initiated homology directed repair. The LoxP sequence is highlighted in yellow along with a novel BamH1 restriction site in red; the underlined text shows the target sequence disrupted by the LoxP sequence/BamH1 site in the ssODN donor. The proximal and distal homology arms are highlighted in blue and gray respectively. (b) Sequence of the 3’ target site in intron 2 of the mouse Plzf allele. The distal and proximal asymmetric homology arms are highlighted in gray and green respectively. The location and sequence of the 3’ target site is highlighted in blue and underlined with the 3’ PAM in red (AGG). Box includes the sequence of the ssODN donor used to insert the 3’ LoxP site into intron 2. The underlined target sequence is disrupted by the LoxP sequence is in yellow and with an additional 3’ BamH1 restriction site in red, which is flanked by the proximal and distal homology arms (green and gray respectively).

FIGURE 2.

FIGURE 2.

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Generation of the mouse Plzff/f conditional knockout allele. (a) Quality scores for the 5’ and 3’ Cas9 guide sequences (sense strand) used to create the murine Plzf conditional knockout allele are shown. Note: in vitro transcribed sgRNAs were microinjected into C57BL/6NJ zygotes. (b) Schematic of the CRISPR/Cas9-mediated targeting strategy to simultaneously insert Lox P sites (open triangles) into intron 1 and 2 to flank Plzf exon 2. The location of the forward and reverse PCR primers to amplify the 5’ and 3’ LoxP sites is indicated. The restriction sites: Xba1, BamH1, Sma1, and EcoR1 are denoted by Xb, B, Sm, and E respectively. Using forward and reverse PCR primers (F1 and R1 to amplify the 5’ LoxP site (200bp) and F2 and R2 to amplify the 3’ LoxP site (200bp)), the gel shows a typical genotype result for wild type (lane 1 (160bp band)), wild type/ LoxP (lane 2 (heterozygote (Plzff/+) 200 and 160bp bands)), and LoxP/LoxP (lane 3 (homozygous (Plzff/f) for the 5’ and 3’ LoxP insertion) 200 bp band)). This PCR result is from genotyping F2 generation mice derived from the F0 #9950 chimeric male mouse. Sequences are shown for 5’ and 3’ LoxP sites carried by these mice (also see Support Information S3).

Thirty-five pups were delivered by ICR dams and were PCR screened for the insertion of the 5’ and 3’ LoxP insertion into the Plzf allele. Table 1 lists the PCR primers used to genotype potential founder pups (F0). For genotyping each LoxP insertion site, one PCR primer in the primer-pair is located outside the sequences of the homology arms in each of the ssODNs; the position of these primers in relation to exon 2 is shown in Support Information Figure S2. From thirty-five pups screened, one potential male founder ((F0) #9950) carried both the 5’ and 3’ LoxP sites. One F0 pup (#9959) was positive for only the 5’ LoxP insertion whereas three pups (F0 #9958; #9960; and #9964) were positive for only the 3’ LoxP insertion (data not shown). To determine whether the 5’ and 3’ LoxP sites in the F0 #9950 mouse are located on the same Plzf allele (in cis) and can pass through the germline, the #9950 mouse was bred with C57BL/6NJ females. Screening progeny from this breeding clearly showed that the 5’ and 3’ LoxP sites are transferred through the germline in cis (Figure 2b). Sanger sequencing of the 5’ LoxP and 3’ LoxP sites using genomic DNA isolated from progeny of the F0 #9950 mouse confirmed that each of the LoxP sites is intact (Support Information Figure S3). Analysis of the top 5 potential off-target sites as scored by the CRISPR Finder tool (https://www.sanger.ac.uk/htgt/wge/) for Cas9 cleavage activity revealed no off-target activity (Support Information Figure S4). Importantly, mice homozygous for the conditional Plzf allele (Plzff/f) are a phenocopy of wild type siblings.

Table 1.

List of PCR primers used for genotyping.

F1 Primer 5’-TTGTGAGCCTCATGGTGGAC-3’
R1 Primer 5’-AAAGGAAGGTTTGCGCTTCC-3’
F2 Primer 5’-AGGGTCTCCTCTGATCTGGA-3’
R2 Primer 5’-TCTGCCTCACAATGTGCCTG-3’
P1 Primer 5’-GAAGATGAGGGCTTGCCCAT-3’
P2 Primer 5’-TTCTCTGCTCTGCAAGGTGG-3’
P3 Primer 5’-GAGATGGCATGAGAAGAGGCT-3’
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To determine whether cre-mediated excision of exon 2 can occur at the Plzff/f allele, we crossed our Plzff/f conditional mouse with a global cre-driver transgenic mouse in which the cytomegalovirus (CMV) promoter drives the expression of cre recombinase (CMVcre) in all cell types (including germ cells) (Schwenk et al., 1995) to generate the Plzfd/d bigenic (Figure 3a). Genotyping with PCR primers: P1, P2, and P3 (Table 1 and Support Information Figure S2), demonstrated absence of exon 2 in the Plzfd/d mouse (Figure 3a, b). Because we have previously demonstrated that Plzf protein is significantly induced in uterine tissue of ovariectomized wild type mice following injection of progesterone hormone (Kommagani et al., 2016), we injected ovariectomized Plzfd/d females and corresponding monogenic controls with either hormone vehicle or progesterone hormone. Western analysis clearly showed that while uterine Plzf protein is not expressed in the vehicle-treated ovariectomized control and Plzfd/d group, Plzf protein expression is only induced in the control uterus six hours following progesterone administration (Figure 3c). The absence of uterine Plzf protein induction by progesterone in the ovariectomized Plzfd/d mouse confirms that cre-mediated excision of exon 2 in the Plzff/f conditional allele results in abrogation of Plzf protein expression.

FIGURE 3.

FIGURE 3.

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Generation of the Plzfd/d bigenic mouse. (a) Schematic which details the breeding scheme to generate the Plzfd/d bigenic mouse. The CMVcre transgenic mouse was crossed with Plzff/f mice to generate the CMVcre: Plzff/f (or Plzfd/d bigenic). The location of the P1, P2, and P3 PCR primers to detect loss of Plzf exon 2 in the Plzfd/d bigenic is shown (also see Support Information Figure S2). (b) Genotyping results using P1 and P2 forward and reverse PCR primers and P1 and P3 forward and reverse PCR primers demonstrate absence of exon 2 in the Plzfd/d bigenic mouse. (c) Western analysis confirms that uterine Plzf protein is not produced in the Plzfd/d mouse. Lanes 1 and 2 represent protein isolated from the uterus of untreated and progesterone-treated ovariectomized wild type mice respectively. Note the significant induction of Plzf protein (75 kDa) expression (after 6-hours) in response to progesterone administration (Kommagani et al., 2016). Lanes 3 and 4 represent uterine protein isolates from untreated and progesterone-treated Plzfd/d mice respectively. Note the clear absence of Plzf protein induction by progesterone in the Plzfd/d mouse (lane 4). Each lane represents uterine protein pooled from four female mice per genotype; the experiment was performed in triplicate (β-actin (43 kDa) served as a loading control).

The tetrapod hindlimb of vertebrates consists of three regions: the proximal stylopod (femur), the intermediate zeugopod (fused tibia and fibula), and the distal autopod (the foot consisting of the tarsals, metatarsals, and phalanges) (Niswander, 2003). As reported for the PlzfKO mouse (Barna et al., 2000), the Plzfd/d bigenic exhibits similar hindlimb abnormalities (Figure 4a, b). Unlike the control (Figure 4c), the Plzfd/d hindlimb develops only rudimentary tibia and fibula structures in the severely shortened zeugopod region (Figure 4d (bracket)). Because of the significant reduction in zeugopod length, the heel bone (calcaneum) and knee are often brought close together, resulting in the plantar surface of the foot twisted away from the ground (Figure 4d), rendering the afflicted hindlimb ineffective for ambulation. While pentadactylyl is the normal digit configuration in tetrapods (Figure 5a), the Plzfd/d bigenic—as previously reported for the PlzfKO mouse (Barna et al., 2000)—exhibits a pre-axial polydactyly foot phenotype, where supernumerary digits (in many cases fused) occur at the anterior margin of the autopod (Figure 5b-e). An additional abnormal skeletal feature of the Plzfd/d mouse is the presence of a kinked tail (Figure 6), a phenotype also reported for the PlzfKO mouse (Barna et al., 2000). Although beyond the scope of the current study, further phenotypic analysis has demonstrated that Plzfd/d male and female mice are infertile (also reported previously (Barna et al., 2000)).

FIGURE 4.

FIGURE 4.

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The Plzfd/d bigenic displays hindlimb abnormalities. (a) Alcian blue and Alizarin red stained skeleton of a monogenic control mouse. (b) Similarly stained skeleton of Plzfd/d mouse. Note the hindlimb defect in the Plzfd/d mouse (arrow and bracket). (c) Larger view of hindlimb of control mouse, showing the femur (stylopod), the patella (knee cap), the fused tibia and fibula (intermediate zeugopod), the calcaneum (heel bone), and the foot (distal autopod); the plantar surface of the foot is indicated by a straight black line. (d) The Plzfd/d hindlimb displays a severe shortening of the zeugopod (bracket), with either an absence or the presence of a rudimentary tibia or fibula. Because the zeugopod region of the Plzfd/d hindlimb is significantly curved, the plantar surface of the foot is twisted away from the ground.

FIGURE 5.

FIGURE 5.

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The Plzfd/d hind foot displays fused digits and polydactylyl. (a) Macro view of hind foot of monogenic control mouse. (b) Similar view of the Plzfd/d hind foot. Note the fusion of three digits (arrowheads and bracket). Compared to control, the Plzfd/d interdigital space is significantly larger (region encompassed by curved bracket). (c-e) Distal autopod skeletal region stained with Alcian blue and Alizarin red. Compared to control (c), note examples of fused digits (arrowhead) and polydactyly in the Plzfd/d hind foot (d and e respectively).

FIGURE 6.

FIGURE 6.

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The Plzfd/d mouse exhibits kinked tails. (a) Macro view of a control tail (1) and Plzfd/d mutant tails (2 and 3). Note the obvious kink in the Plzfd/d tail (arrowhead). (b-d) Corresponding Alcian blue and Alizarin red stained tailbone structure from control (c) and Plzfd/d mutant mice (c and d); arrowheads indicate location of the tail kink.

In conclusion, we have successfully generated a new conditional knockout Plzff/f mouse model using CRISPR/Cas9 genome editing. We predict that the Plzff/f mouse will serve as an invaluable tool with which to further elucidate Plzf function in a tissue and cell-type specific manner. For example, we recently demonstrated that PLZF is essential for human endometrial stromal cell decidualization (Kommagani et al., 2016; Szwarc et al., 2018), a cellular process required for embryo implantation. Crossing the Plzff/f mouse with our previously reported progesterone receptor promoter-driven cre (Pgrcre) knockin mouse (Soyal et al., 2005) will now allow us to functionally validate Plzf action specifically in the mouse endometrium, without interference of phenotypes contributed by other target tissues due to this transcription factor’s loss-of-function.

3. MATERIALS AND METHODS

3.1. Generation of the Plzff/f mouse using CRISPR/Cas9 gene editing

Two separate sgRNAs and two ssODNs with LoxP sequences were used to insert (or knock-in) a LoxP site 5’ and 3’ of exon 2 of the mouse Plzf gene. Encoding more than 50% of murine Plzf protein, exon 2 of the Plzf gene was selected based on the targeting strategy previously reported by the Pandolfi group (Barna et al., 2000) and on the conditional knockout vector design detailed by the International Mouse Phenotyping Consortium/Knockout Mouse Project (IMPC/KOMP (Zbtb16 tm405544a(L1L2_Bact_P); www.informatics.jax.org/marker/MGI: 103222)). Established methods were used to co-microinject 100ng/μl Cas9 mRNA, 20ng/μl validated sgRNA (each) and 100ng/μl of ssODNs (each) into the cytoplasm of 200 C57BL/6NJ embryos (Lanza et al., 2018). Following micro-injection, zygotes were transferred into pseudopregnant ICR recipient females at approximately 25–32 zygotes per recipient. Both C57BL/6NJ and ICR mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Established methods of genotyping potential founder mice were followed (Lanza et al., 2018). Also, Sanger sequencing of cloned LoxP sites from mouse genomic DNA described previously were followed in these studies (Lanza et al., 2018).

3.2. Analysis of off-target Cas9 activity

The top five potential off-target sites for each sgRNA in the conditional targeting of the mouse Plzf gene were identified using the Wellcome Trust Sanger Institute Genome Editing website (http://www.sanger.ac.uk/htgt/wge/). Flanking PCR primers designed to amplify 80–180 base pair amplicons are listed in Support Information Figure S4 with the location, sequence, and number of mismatches from the original sgRNA. Genomic DNA from F2 mice was prepared as previously described (Lanza et al., 2018) and assayed by High Resolution Melt Analysis using the MeltDoctor HRM Master Mix (ThermoFisher Scientific Inc., Waltham, MA (#4415440)) according to the manufacturer’s protocol and run on the Applied Biosystems QuantStudio 7 Flex Real-Time PCR System (ThermoFisher Scientific Inc.). At least two wild-type samples were analyzed concurrently with the test samples. For any sample with a HRM analysis result deviating from the wild-type sample, suggesting a mutagenesis event, PCR products were cloned and Sanger sequenced as described above.

3.2. Generation of the Plzfd/d bigenic mouse

To generate the Plzfd/d bigenic mouse, the Plzff/f mouse was crossed with the CMVcre mouse in the C57/BL6 inbred strain (The Jackson Laboratory, Bar Harbor, ME (stock number: 006054; strain name B6.C-Tg(CMV-cre)1 Cgn/J (Schwenk et al., 1995))). Through cre-mediated excision in this bigenic, the floxed Plzf gene is ablated in all Plzfd/d tissues, including germ cells. Note: in the CMVcre mouse, the cre gene is X-linked and therefore transgene transmission through males is restricted to female progeny.

Mice were housed in an AAALAC accredited vivarium at Baylor College of Medicine. In temperature-regulated rooms (22 ± 2oC) with a cycling 12-hour lights-on:12-hour lights-off schedule, mice were fed irradiated Tekland global soy protein-free extruded rodent diet (Harlan Laboratories, Inc., Indianapolis, IN) and fresh water ad libitum. Pre-approved experimental protocols applied to mice were conducted in accordance with the guidelines described in the Guide for the Care and Use of Laboratory Animals (“The Guide” (Eighth Edition 2011)), published by the National Research Council of the National Academies, Washington, D.C. (www.nap.edu). For all animal procedures described in this study, prior approval from the Institutional Animal Care and Use Committee (IACUC) at Baylor College of Medicine was obtained. As with our previous mouse models, the Plzff/f mouse will be made freely available to the scientific community upon request.

3.4. Whole-mount skeletal staining

To assess skeletal patterning, an established Alcian blue and Alizarin red staining protocol was followed (Rigueur and Lyons, 2014). Following euthanasia, skin, internal organs, and brown fat were removed from monogenic control and Plzfd/d mice. After a rinse with 1XPBS, tissues were dehydrated and fixed in two changes of 95% ethanol over a 16-hour period. Tissues were then placed in 100% acetone for further fixation and removal of adipose tissue. Following 48 hours of acetone incubation, Tissues were transferred to 0.03% Alcian blue solution (dissolved in 80% ethanol and 20% glacial acetic acid) for 24 hours to stain cartilage. For de-staining purposes, tissues were washed in two changes of 70% ethanol before incubation in 95% ethanol overnight. Following ethanol washes, soft tissue was hydrolyzed in 1% potassium hydroxide (KOH dissolved in distilled water) overnight to pre-clear the tissue and allow visualization of stained skeletal structures. Skeletal specimens were then incubated in 0.005% Alizarin red solution (in 1% KOH) for 2–5 days for bone staining. Following bone staining, skeletal specimens were transferred to a 1% KOH clearing solution. For long-term storage, stained skeletons were stored in 100% glycerol.

3.5. Hormone treatment and immunoblot analysis

To assess uterine Plzf protein induction by short-term progesterone exposure, female mice were ovariectomized at six weeks-of-age and rested for two weeks to void remaining circulating levels of ovarian steroid hormones (Kommagani et al., 2016). Following two weeks of rest, ovariectomized mice were then subcutaneously injected with 1mg of progesterone (Sigma-Aldrich, St. Louis, MO; in 100μl sesame oil) or 100μl vehicle. Mice were euthanized six hours later before uteri were removed for protein isolation using standard approaches (Kommagani et al., 2016). As detailed previously (Kommagani et al., 2016), the following primary antibodies were used for western analysis: a rabbit polyclonal anti-Plzf H-300 (Santa Cruz Biotechnology Inc., Dallas, TX (# SC-22839)) and a mouse monoclonal anti-β−actin AC-74 (Sigma-Aldrich; #A2228). Corresponding horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology Inc.) were used as secondary antibodies and the chemiluminescent signal was visualized using the SuperSignal West Pico Chemiluminescent Substrate Kit (ThermoFischer Scientific Inc., #34580).

Supplementary Material

Supp figS1

Support Information Figure S1. The location of the 5’ and 3’ Cas9 guide sequences are highlighted in green and blue respectively. Exon 2 of the mouse Plzf gene is highlighted in yellow.

Supp figS2

Support Information Figure S2. The location of the PCR primers to genotype mice for insertion of the 5’ and 3’ LoxP sites (F1/R1 and F2/R2 respectively) and to genotype loss of exon 2 (P1, P2, and P3) are shown. Sequences of these primers are listed in Table 1.

Supp figS3

Support Information Figure S3. Screen shots of the region encompassing the 5’ and 3’ LoxP sequences (blue) inserted in the intron 1 and 2 of the mouse Plzf gene respectively. Mutations were not found in the CRISPR/Cas9-mediated LoxP insertions into the mouse Plzf allele (sequence track 3 compared to sequence track 1). Sequence alignments were performed using SnapGene software (Chicago, IL).

Supp figS4

Support Information Figure S4. Off-target Cas9 activity was analyzed by high resolution meltanalysis. The top 5 potential off-target sites for the 5’ and the 3’ sgRNA used in the conditional targeting of the murine Plzf gene were identified using the WTSI Genome Editing website (https://www.sanger.ac.uk/htgt/wge). Flanking PCR primers designed to amplify 80-180 bp amplicons are listed. The location, sequence, and number of mismatches from the original sgRNA are shown. As described in the Material and Methods section, off-target mutagenesis was assessed by High Resolution MeltAnalysis using MeltDoctor HRM Master Mix on the QuantStudio 7 Flex Real-Time PCR system. At least three wild-type samples were analyzed concurrently with the test samples.

ACKNOWLEDGMENTS

For the invaluable technical assistance provided, the authors thank Jie Li, Yan Ying, and Rong Zhao. The authors also thank the Mouse Embryonic Stem Cell and Genetically Engineered Mouse advanced technical cores at Baylor College of Medicine for their technical expertise. Technical cores were funded in part by the National Institutes of Health Cancer Center Grant (P30 CA125123) and by the Knockout Mouse Project (KOMP3) grant (U42 HG0063521). Finally, this work was supported in part by National Institutes of Health/National Institute of Child and Human Development RO1 grant: HD042311 to JPL.

REFERENCES

  1. Barna M, Hawe N, Niswander L, Pandolfi PP. 2000. Plzf regulates limb and axial skeletal patterning. Nat Genet 25: 166–172. [DOI] [PubMed] [Google Scholar]
  2. Barna M, Pandolfi PP, Niswander L. 2005. Gli3 and Plzf cooperate in proximal limb patterning at early stages of limb development. Nature 436: 277–281. [DOI] [PubMed] [Google Scholar]
  3. Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE. 2004. Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet 36: 647–652. [DOI] [PubMed] [Google Scholar]
  4. Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M, Orwig KE, Wolgemuth DJ, Pandolfi PP. 2004. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 36: 653–659. [DOI] [PubMed] [Google Scholar]
  5. Fahnenstich J, Nandy A, Milde-Langosch K, Schneider-Merck T, Walther N, Gellersen B. 2003. Promyelocytic leukaemia zinc finger protein (PLZF) is a glucocorticoid- and progesterone-induced transcription factor in human endometrial stromal cells and myometrial smooth muscle cells. Mol Hum Reprod 9: 611–623. [DOI] [PubMed] [Google Scholar]
  6. Fernandez-Valdivia R, Jeong J, Mukherjee A, Soyal SM, Li J, Ying Y, Demayo FJ, Lydon JP. 2010. A mouse model to dissect progesterone signaling in the female reproductive tract and mammary gland. Genesis 48: 106–113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fischer S, Kohlhase J, Bohm D, Schweiger B, Hoffmann D, Heitmann M, Horsthemke B, Wieczorek D. 2008. Biallelic loss of function of the promyelocytic leukaemia zinc finger (PLZF) gene causes severe skeletal defects and genital hypoplasia. J Med Genet 45: 731–737. [DOI] [PubMed] [Google Scholar]
  8. Gaber ZB, Butler SJ, Novitch BG. 2013. PLZF regulates fibroblast growth factor responsiveness and maintenance of neural progenitors. PLoS Biol 11: e1001676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Green M 1955. Luxoid-A new hereditary leg and foot abnormality: In the house mouse. Journal of Heredity 46: 91–99. [Google Scholar]
  10. Hashimoto-Partyka MK, Lydon JP, Iruela-Arispe ML. 2006. Generation of a mouse for conditional excision of progesterone receptor. Genesis 44: 391–395. [DOI] [PubMed] [Google Scholar]
  11. Ikeda R, Yoshida K, Tsukahara S, Sakamoto Y, Tanaka H, Furukawa K, Inoue I. 2005. The promyelotic leukemia zinc finger promotes osteoblastic differentiation of human mesenchymal stem cells as an upstream regulator of CBFA1. J Biol Chem 280: 8523–8530. [DOI] [PubMed] [Google Scholar]
  12. Kommagani R, Szwarc MM, Vasquez YM, Peavey MC, Mazur EC, Gibbons WE, Lanz RB, DeMayo FJ, Lydon JP. 2016. The Promyelocytic Leukemia Zinc Finger Transcription Factor Is Critical for Human Endometrial Stromal Cell Decidualization. PLoS Genet 12: e1005937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kovalovsky D, Uche OU, Eladad S, Hobbs RM, Yi W, Alonzo E, Chua K, Eidson M, Kim HJ, Im JS, Pandolfi PP, Sant’Angelo DB. 2008. The BTB-zinc finger transcriptional regulator PLZF controls the development of invariant natural killer T cell effector functions. Nat Immunol 9: 1055–1064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kren V 1975. Genetics of the polydactyly-luxate syndrome in the Norway rat, Rattus norvegicus. Acta Univ Carol Med Monogr: 1–103. [PubMed] [Google Scholar]
  15. Labbaye C, Quaranta MT, Pagliuca A, Militi S, Licht JD, Testa U, Peschle C. 2002. PLZF induces megakaryocytic development, activates Tpo receptor expression and interacts with GATA1 protein. Oncogene 21: 6669–6679. [DOI] [PubMed] [Google Scholar]
  16. Lanza DG, Gaspero A, Lorenzo I, Liao L, Zheng P, Wang Y, Deng Y, Cheng C, Zhang C, Seavitt JR, DeMayo FJ, Xu J, Dickinson ME, Beaudet AL, Heaney JD. 2018. Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles. BMC Biol 16: 69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liska F, Peterkova R, Peterka M, Landa V, Zidek V, Mlejnek P, Silhavy J, Simakova M, Kren V, Starker CG, Voytas DF, Izsvak Z, Pravenec M. 2016. Targeting of the Plzf Gene in the Rat by Transcription Activator-Like Effector Nuclease Results in Caudal Regression Syndrome in Spontaneously Hypertensive Rats. PLoS One 11: e0164206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liu TM, Lee EH, Lim B, Shyh-Chang N. 2016. Concise Review: Balancing Stem Cell Self-Renewal and Differentiation with PLZF. Stem Cells 34: 277–287. [DOI] [PubMed] [Google Scholar]
  19. Mao AP, Constantinides MG, Mathew R, Zuo Z, Chen X, Weirauch MT, Bendelac A. 2016. Multiple layers of transcriptional regulation by PLZF in NKT-cell development. Proc Natl Acad Sci U S A 113: 7602–7607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Niswander L 2003. Pattern formation: old models out on a limb. Nat Rev Genet 4: 133–143. [DOI] [PubMed] [Google Scholar]
  21. Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE. 2016. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 34: 339–344. [DOI] [PubMed] [Google Scholar]
  22. Rigueur D, Lyons KM. 2014. Whole-mount skeletal staining. Methods Mol Biol 1130: 113–121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Savage AK, Constantinides MG, Bendelac A. 2011. Promyelocytic leukemia zinc finger turns on the effector T cell program without requirement for agonist TCR signaling. J Immunol 186: 5801–5806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Savage AK, Constantinides MG, Han J, Picard D, Martin E, Li B, Lantz O, Bendelac A. 2008. The transcription factor PLZF directs the effector program of the NKT cell lineage. Immunity 29: 391–403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schwenk F, Baron U, Rajewsky K. 1995. A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res 23: 5080–5081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Singh SP, Zhang HH, Tsang H, Gardina PJ, Myers TG, Nagarajan V, Lee CH, Farber JM. 2015. PLZF regulates CCR6 and is critical for the acquisition and maintenance of the Th17 phenotype in human cells. J Immunol 194: 4350–4361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Soyal SM, Mukherjee A, Lee KY, Li J, Li H, DeMayo FJ, Lydon JP. 2005. Cre-mediated recombination in cell lineages that express the progesterone receptor. Genesis 41: 58–66. [DOI] [PubMed] [Google Scholar]
  28. Suliman BA, Xu D, Williams BR. 2012. The promyelocytic leukemia zinc finger protein: two decades of molecular oncology. Front Oncol 2: 74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Szwarc MM, Hai L, Gibbons WE, Peavey MC, White LD, Mo Q, Lonard DM, Kommagani R, Lanz RB, DeMayo FJ, Lydon JP. 2018. Human endometrial stromal cell decidualization requires transcriptional reprogramming by PLZF. Biol Reprod 98: 15–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vincent-Fabert C, Platet N, Vandevelde A, Poplineau M, Koubi M, Finetti P, Tiberi G, Imbert AM, Bertucci F, Duprez E. 2016. PLZF mutation alters mouse hematopoietic stem cell function and cell cycle progression. Blood 127: 1881–1885. [DOI] [PubMed] [Google Scholar]
  31. Wang N, Frank GD, Ding R, Tan Z, Rachakonda A, Pandolfi PP, Senbonmatsu T, Landon EJ, Inagami T. 2012. Promyelocytic leukemia zinc finger protein activates GATA4 transcription and mediates cardiac hypertrophic signaling from angiotensin II receptor 2. PLoS One 7: e35632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Xu D, Holko M, Sadler AJ, Scott B, Higashiyama S, Berkofsky-Fessler W, McConnell MJ, Pandolfi PP, Licht JD, Williams BR. 2009. Promyelocytic leukemia zinc finger protein regulates interferon-mediated innate immunity. Immunity 30: 802–816. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp figS1

Support Information Figure S1. The location of the 5’ and 3’ Cas9 guide sequences are highlighted in green and blue respectively. Exon 2 of the mouse Plzf gene is highlighted in yellow.

NIHMS1013296-supplement-Supp_figS1.pdf (1.1MB, pdf)
Supp figS2

Support Information Figure S2. The location of the PCR primers to genotype mice for insertion of the 5’ and 3’ LoxP sites (F1/R1 and F2/R2 respectively) and to genotype loss of exon 2 (P1, P2, and P3) are shown. Sequences of these primers are listed in Table 1.

NIHMS1013296-supplement-Supp_figS2.pdf (1.8MB, pdf)
Supp figS3

Support Information Figure S3. Screen shots of the region encompassing the 5’ and 3’ LoxP sequences (blue) inserted in the intron 1 and 2 of the mouse Plzf gene respectively. Mutations were not found in the CRISPR/Cas9-mediated LoxP insertions into the mouse Plzf allele (sequence track 3 compared to sequence track 1). Sequence alignments were performed using SnapGene software (Chicago, IL).

NIHMS1013296-supplement-Supp_figS3.pdf (3.4MB, pdf)
Supp figS4

Support Information Figure S4. Off-target Cas9 activity was analyzed by high resolution meltanalysis. The top 5 potential off-target sites for the 5’ and the 3’ sgRNA used in the conditional targeting of the murine Plzf gene were identified using the WTSI Genome Editing website (https://www.sanger.ac.uk/htgt/wge). Flanking PCR primers designed to amplify 80-180 bp amplicons are listed. The location, sequence, and number of mismatches from the original sgRNA are shown. As described in the Material and Methods section, off-target mutagenesis was assessed by High Resolution MeltAnalysis using MeltDoctor HRM Master Mix on the QuantStudio 7 Flex Real-Time PCR system. At least three wild-type samples were analyzed concurrently with the test samples.

NIHMS1013296-supplement-Supp_figS4.pdf (963.1KB, pdf)

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