Molecular Vision 2016; 22:705-717 <>
Received 05 August 2015 | Accepted 21 June 2016 | Published 23 June 2016

Pou4f2 knock-in Cre mouse: A multifaceted genetic tool for vision researchers

Aaron B. Simmons,1 Samuel J. Bloomsburg,1 Samuel A. Billingslea,1 Morgan M. Merrill,1 Shuai Li,1 Marshall W. Thomas,2 Peter G. Fuerst1,3

1Department of Biological Sciences, University of Idaho, Moscow, ID; 2Department of Biology, Brigham Young University—Idaho, Rexburg, ID; 3WWAMI Medical Education Program, University of Washington School of Medicine, Moscow, ID

Correspondence to: Peter G. Fuerst, Department of Biological Sciences, University of Washington WWAMI Medical Education Program, University of Idaho, Moscow, Idaho 83844; Phone: (208) 885 7512 ; FAX: (208) 885 7905; email:


Purpose: A transgenic mouse that expresses Cre recombinase under control of the Pou4f2-promoter (also referred to as Brn-3b and Brn-3.2) was characterized. Pou4f2 expression has been reported in a subset of retinal ganglion cells (RGCs) in the retina, in the midbrain, and in the germline. In this study, we characterize the expression pattern of this Cre-recombinase line and report its utility in targeted deletion, temporal deletion, RGC depletion, and germline targeting, which can be regulated by the sex of the Cre-carrying mouse.

Methods: Pou4f2Cre was mapped by using a combination of PCR and sequencing of PCR products to better understand the construct and to locate where it was inserted within the Pou4f2 locus. Cre expression patterns were examined by crossing Pou4f2Cre/+ mice to Cre reporter mice. Immunohistochemistry was used to further define the pattern of Cre expression and Cre-mediated recombination within the retina, brain, and other tissues.

Results: An internal ribosome entry site (IRES)-Cre cassette was inserted into the Pou4f2 gene disrupting normal gene function, as verified by the depletion of RGCs in mice homozygous for the insert. Pou4f2Cre expression was observed in the retina, brain, peripheral neurons, and male germ cells. Germline recombination was observed when the sire carried the Cre and the target for recombination. In all other breeding schemes, recombination was observed within subsets of cells within the retina, brain, intestines, heart, and gonads. In the retina, Cre efficiently targets recombination in neurons within the RGC layer (RGL), the inner nuclear layer (INL), and a small percentage of photoreceptors, activity that has not been previously reported. Unlike most other Cre lines active in the inner retina, recombination in Müller and other glia was not observed in mice carrying Pou4f2Cre. Within the visual centers of the brain, Cre targets recombination in about 15% of cells within the superchiasmatic nucleus, lateral geniculate nucleus, and superior colliculus.

Conclusions: Pou4f2Cre provides multiple uses for the vision researcher’s genetic toolkit. First, Pou4f2Cre is a knock-in allele that can be used to eliminate Pou4f2, resulting in depletion of RGCs. Second, expression of Cre in male germ cells makes this strain an efficient germline activator of recombination, for example, to target LoxP-flanked sequences in the whole mouse. Third, Pou4f2Cre efficiently targets RGCs, amacrine cells, bipolar cells, horizontal cells, and a small number of photoreceptors within the retina, as well as the visual centers in the brain. Unlike other Cre recombinase lines that target retinal neurons, no recombination was observed in Müller or other retinal glia. These properties make this Cre recombinase line a useful tool for vision researchers.


Targeted manipulation of the genome has greatly improved our understanding of the mechanisms responsible for development; however, many such genetic manipulations result in severe abnormalities and high mortality rates. Understanding cell autonomy of defects often also necessitates a more targeted approach than eliminating or overexpressing a gene. Cre-lox technology, in which Cre recombinase excises or inverts the sequence flanked by LoxP sites, depending on the orientation of the sites, allows for targeted activation or inactivation of DNA sequences [1]. Using this technology has enabled researchers to target specific populations of cells or specific time points. Characterizing and disseminating various Cre transgenic lines allows researchers to refine experiments and ask better informed questions.

In this study, we characterize a mouse strain in which Cre recombinase has been inserted into the Pou4f2 locus (also referred to as Brn-3b or Brn-3.2), replacing the Pou4f2 gene. Previous work has shown that the POU domain transcription factor Pou4f2 is expressed in a large subset of retinal ganglion cells (RGCs) and is essential for RGC differentiation [2-5], survival [6] and axon path-finding [7-9]. The study herein characterizes a Pou4f2 knock-in Cre recombinase mouse line, Pou4f2Cre, which expresses Cre recombinase in a subset of retinal neurons. In addition to targeting genes in the mouse retina, the Pou4f2Cre line can be used to generate mice in which the number of RGCs is depleted, when the transgene is homozygous [2,3]. When carried by male mice, along with a target allele, Pou4f2Cre efficiently targets recombination in the germline. Therefore, a single mouse strain can be used to target recombination in retinal neurons, to deplete RGCs, and to target germline recombination.


Mouse care and housing

All protocols were performed in accordance with the University of Idaho Institutional Animal Care and Use Committee and with The Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were fed ad libitum under a 12 h:12 h light-dark cycle.

Mouse strains

The following mouse strains were used in this study: Pou4f2Cre (courtesy of Dr. Vann Bennett; Duke University) [10], Ai9 reporter (The Jackson Laboratory, Bar Harbor, ME; stock number 007909) [11], Brainbow reporter (The Jackson Laboratories stock number 021,227) [12], and Dscam conditional mutant mice [10]. Mouse strains were maintained on a mixed C57Bl6/J, C3H/HeJ, and 129 background. All mice carried the wild-type allele of Pde6b.


Tissue biopsies (toe tips or tail tips) were taken from the mice and then prepared for PCR by first boiling the samples in 50 μl of 25 μM sodium hydroxide and 0.2 μm EDTA for 15 min. Fifty microliters of Tris Cl (pH 5.0) was then added to the samples, neutralizing them. Each PCR reaction consisted of 7.5 μl of OneTaq Hot Start Master Mix (2X, New England Biolabs, Ipswich, MA) [13], 0.1 μl primers, 1 μl of DNA sample, and 6.4 μl of nuclease-free water, bringing the final volume to 15 μl. PCR conditions: (36 cycles; 94 °C 30 s, 57 °C 30 s, 72 °C 2 min). The following primers were used when genotyping: Pou4f2 forward (#1, Figure 1A): 5′-GCT GAG GTC TGA AGC CAG AG-3′, Pou4f2 reverse (#2, Figure 1A): 5′-CAG TCA GCT CCT CGC TTT CT-3′; Cre forward (#3, Figure 1B): 5′-GCA TTA CCG GTC GAT GCA ACG AGT G-3′, Cre reverse (#4, Figure 1B): 5′-GAG TGA ACG AAC CTG GTC GAA ATC A-3′; and neomycin forward (#6, Figure 1B): 5′-AGG ATC TCC TGT CAT CTC ACC TTG CTC CTG-3′, neomycin reverse (#5, Figure 1B): 5′-AAG AAC TCG TCA AGA AGG CGA TAG AAG GCG-3′.

Tissue preparation

Mice were anesthetized with an intraperitoneal injection of tribromoethanol (500 mg per kg body weight). Anesthetized mice were perfused with PBS (1X; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4; 1.8 mM KPO4), pH 7.4. The eyes were then carefully removed using forceps and dissected in PBS to remove the cornea, iris, and lens. Eyes were fixed in 4% paraformaldehyde (PFA) solution for 30 min at room temperature, and the retina was dissected from the eyecup. Fixed retinas were sunk in 30% sucrose overnight before freezing. Sucrose sunk tissues were placed in Tissue-Tek optimal cutting temperature (OCT) media (Sakura Finetek, Torrance, CA) and frozen by placing the block above liquid nitrogen to rapidly freeze the sample. Retinas were then sectioned at 10 μm and placed onto charged slides. Whole fixed retinas were washed thoroughly in PBS and then stained. All other tissues were fixed in 4% PFA overnight at 4 °C and washed thoroughly with PBS. Tissues for cryosectioning were handled in the same manner as the retinas. Brains that were used for the vibratome sections were placed in a 6% agarose block, sectioned at 150 μm thickness, and stained with 4,6-diamidino-2-phenylindole (DAPI) reagent at a dilution of 1:50,000 of 1 mg/ml stock.


Sections-- Tissues were blocked in a blocking solution (5% normal donkey serum, 0.1% Triton X-100, and PBS) for 30 min at room temperature. Blocking solution was used to dilute the primary antibodies. Sections were incubated in primary antibodies overnight at 4 °C. Sections were then washed in PBS at room temperature three times before secondary staining. Secondary antibodies were also diluted in blocking solution and incubated overnight at 4 °C. Sections were again washed three times in PBS at room temperature. DAPI was added to the second wash at a concentration of 1:50,000 from 1 mg /ml stock. Coverslips were applied using 80% glycerol as a mounting medium.

Whole retinas-- Whole retinas were stained in a similar manner except the blocking solution used was composed of 5% normal donkey serum, 0.4% Triton X-100, and PBS, blocking was performed at 4 °C overnight. Both primary and secondary incubations lasted 2–4 days at 4 °C, and the final wash was performed in 0.4% Triton blocking solution overnight at 4 °C.


The following antibodies were used in this study: mouse anti-POU4F1/BRN3A (Millipore, Darmstadt, Germany; MAB1585; 1:100); goat anti-POU4F2/BRN3B (Santa Cruz Biotechnology, Santa Cruz, CA; sc-6026; 1:100); rabbit anti-melanopsin (generous gift from Ignacio Provencio; 1:10,000); rabbit anti-tyrosine hydroxylase (Millipore; AB152; 1:500); mouse anti-AP2α (Developmental Studies Hybridoma Bank, Trevor J. Williams; 3B5; 1:50); rabbit anti-calbindin (Swant, Marly, Switzerland; CB-38a; 1:1,000); goat anti-choline acetyltansferase (Millipore; AB144P; 1:250); rabbit anti-bNOS (Sigma, St. Louis, MO; N7280; 1:5,000); mouse anti-PKCα (Santa Cruz Biotechnology, sc-17769; 1:500); rabbit anti-cone arrestin (Millipore; AB15282; 1:10,000); mouse anti-glutamine synthetase (Millipore; MAB302; 1:1,000); mouse anti-calsenilin (Millipore; 05–756; 1:1,000); mouse anti-PAX6 (developed by Kawakami, Developmental Studies Hybridoma; 1:500); and rabbit anti-Cre (Millipore; 69,050-3; 1:500). All secondary antibodies were used at 1:1,000 (Jackson ImmunoResearch, West Grove, PA).

Fluorescent microscopy

Sections and whole retinas were imaged using an Olympus (Center Valley, PA) or Nikon (Melville, NY) spinning disk confocal microscope or an Olympus FluoView confocal microscope. Adobe Photoshop (CS6; San Jose, CA) was used to crop and rotate the images. If brightness or contrast was altered, it was done uniformly across the entire image.


Pou4f2Cre can be used to deplete RGCs

The Pou4f2Cre transgenic line used in this study was previously generated but never characterized [10]. As the method used to generate the mice was not known, numerous primers were designed specific to the native Pou4f2 sequence and Cre recombinase to determine the site of Cre insertion within the Pou4f2 locus (Figure 1A,B and not shown). Additionally, primers designed specifically for neomycin were created to determine whether this cassette still remained within the transgene (Figure 1B). Through the use of PCR and sequencing of the PCR products, we successfully mapped the location of Pou4f2Cre (Figure 1C and not shown). The inserted transgene consists of an internal ribosome entry site (IRES) Cre fused to exon 1 of the native Pou4f2 sequence and an inverted neomycin cassette (Figure 1B). Homozygous carriers of the transgene can be differentiated from heterozygotes by the lack of the Pou4f2 gene, assayed with PCR (Figure 1D). Elimination of POU4F2 (BRN3B) immunoreactivity in the retina and a reduction in RGCs, assayed with an antibody to the homologous protein POU4F1 (BRN3A), was observed in mice homozygous for Cre recombinase, consistent with previous investigations of Pou4f2 function [2,3] (Figure 1E–G).

Location of Pou4f2Cre recombination is dependent upon breeding scheme

To test the efficiency and location of Pou4f2Cre recombination activity, Pou4f2Cre mice were crossed to a mouse strain carrying a sensitive and ubiquitously expressed Cre reporter, Ai9 [11]. The Ai9 reporter (Figure 2A) is driven by the CAG promoter, which is followed by a LoxP-flanked transcription termination cassette and red fluorescent protein (RFP). Upon Cre-mediated recombination, the transcription termination cassette is removed, allowing for the transcription of RFP. This results in the production of RFP in tissues that have expressed Cre recombinase.

Cre activity was determined by visualization of RFP in cryosections and tissue biopsies of mouse tissues (Figure 2C–E). The pattern of recombination was dependent upon which parent carried the Cre and the target of Cre. When the sire carried Pou4f2Cre and Ai9, germline recombination occurred (Figure 2B,D). Germline recombination of the reporter was observed in offspring regardless of whether they inherited Pou4f2Cre, verified with PCR (not shown), suggesting that recombination was targeted in the germline. RFP was observed within the testis of sexually mature mice that carried Pou4f2Cre and the Ai9 reporter (Figure 2E), consistent with these results and previous reports that Pou4f2 is expressed within germ cells [14,15]. When the sire carried Pou4f2Cre and the dam carried the reporter, recombination was limited to subsets of cells within the retina, brain, intestines, heart, and gonads (Figure 2Bii,E). When the dam carried Pou4f2Cre, recombination was limited to subsets of cells within the retina, brain, intestines, heart, and gonads, regardless of which parent carried the reporter allele (Figure 2Biii–iv,E). No visualization of the Ai9 reporter was detected in the mice that lacked Pou4f2Cre (Figure 2C).

Pou4f2Cre-mediated recombination within the retina

To test the specificity of Pou4f2Cre expression within the retina, Pou4f2Cre/+ Ai9 retinal sections from crosses generating specific recombination (Figure 2Bii–iv,E) were immunostained with a panel of cell-type specific markers (Figure 3, Figure 4, and Table 1). Reporter activity was limited to neurons but was also detected in neuron populations not known to express Pou4f2. Recombination was observed in RGCs (Figure 3A,B), amacrine cells (ACs; Figure 3C–E and Figure 4A–B), bipolar cells (BCs; Figure 4C), horizontal cells (HCs; Figure 3D), and a very small number of photoreceptors (Figure 4D). No Cre activity was detected within the microglia, astrocytes, or blood vessels (not shown). Müller glia were also not normally targeted by Cre (Figure 4E,E’’). However, one of the 34 mice observed in this study contained columns of cells that produced RFP, suggesting that a small number of retinal progenitor cells had expressed Cre (not shown). For the majority of markers used in this study, only a portion of the cells produced RFP, with the exception of POU4F2, where all counted cells produced RFP (Table 1). Pou4f2Cre/+ mice were also crossed with mice carrying the Brainbow reporter (Figure 4F). The Brainbow reporter is a transgene driven by the Thy1 promoter that encodes a series of fluorescent proteins, each flanked by LoxP sites [16]. This genetic construct allows for differential expression and visualization of fluorescent proteins. Cre expression within the retina was again limited to neurons within the retina but only those that express Thy1: RGCs, ACs, and HCs (Figure 4F).

Pou4f2Cre/+ Ai9 retinal sections were taken from crosses that generated a specific recombination to map out the developmental time course at which Pou4f2Cre targeted recombination in different cell populations (Figure 2Bii–iv,E). The proportion of cells producing RFP and positive for specific markers were quantified at P0, P10, P20, and P40 (Figure 5). The proportion of cells positive for RFP and PAX6, ChAT, and calsenilin increased incrementally from P0 to P40, while POU4F2-positive cells were all targeted starting from P0, the earliest time point sampled (Figure 5A–I). Immunohistochemistry was then used to detect the location of the Cre protein (Figure 5J–M). The Cre protein was observed within the neurites and nuclei of cells in which recombination occurred (Figure 5J), while staining was absent in retinal sections taken from mice that lacked Cre (Figure 5K). The Cre protein was observed within the nuclei and neurites of subsets of ganglion cells and horizontal cells, confirmed by costaining with POU4F2 and PAX6 (not shown) but was absent from the nuclei of amacrine and bipolar cells at P10 (Figure 5L,L’). The Cre protein was observed within the nuclei of cells in the RGL and the INL by P40, consistent with reporter activation (Figure 5M,M’). Late activity of Pou4f2Cre was also effective in targeting constructs other than the highly sensitive Ai9 and Brainbow reporters. A conditional allele of Down syndrome cell adhesion molecule (Dscam) was used to demonstrate this activity, which is evidenced by the accumulation of DSCAM protein with the cells bodies of targeted cells [17]. Dscam targeting was observed in ganglion cells and amacrine cells early during development and later (about P45) in bipolar cells (Figure 5N).

Pou4f2Cre activity within the brain

Pou4f2Cre/+ Ai9 brains from crosses that generated a specific recombination were imaged to test the specificity of Pou4f2Cre expression within the brain (Figure 2Bii–iv,E). RFP was observed in many regions of the brain (Figure 6). The expression of RFP was observed in retinal ganglion cell axons projecting throughout the brain but was also found within neural cell bodies and their neurites in other regions of the brain, including the visual centers, consistent with previous studies [9,18]. The percentage of cells targeted by Pou4f2Cre within three visual centers was determined: the suprachiasmatic nucleus (SCN), the lateral geniculate nucleus (LGN), and the superior colliculus (SC; Figure 7). This was accomplished by staining vibratome sections of the brain with DAPI and counting the total number of cells positive for RFP. Our results indicate that 18% of cells within the SCN had expressed Cre, 11% in the LGN, and 17% in the SC (Figure 7D-G).


This study characterized a Pou4f2Cre transgenic mouse line, in which Cre recombinase is driven by the Pou4f2 promoter. Our results show that this transgenic mouse can be used (1) to deplete RGC populations when homozygous for the Pou4f2Cre, (2) that the pattern of Cre recombination is dependent upon which parent carries Pou4f2Cre and the target of recombination, making it useful to target germline recombination, (3) that Cre specific activity within the retina is limited to the neurons (RGCs, ACs, BCs, HCs, and a small number of photoreceptors), and (4) that the timing of Pou4f2Cre recombination is subclass specific. These properties make this line a useful multipurpose genetic reagent for vision researchers.

The Pou4f2Cre knock-in transgenic mouse can be used to generate RGC-specific depletion, consistent with previous literature demonstrating that deletion of Pou4f2 results in the depletion of about 70% of the RGCs [2,3]. Previous studies have also shown that the remaining RGCs within Pou4f2 knockout mice maintain normal RGC diversity and a grossly normal morphology [19]. Using the Pou4f2Cre transgenic mouse line to drastically reduce RGC populations allows questions to be asked about how retinas develop without normal populations of RGCs and the effects major depletion of RGCs has on the developing retina.

The Pou4f2Cre line can also be used to target recombination in the germline, depending on which parent carries the Pou4f2Cre and the target for recombination. Germline recombination occurs when the sire carried the Pou4f2Cre gene and the target for recombination yet was always specific when the dam carried Pou4f2Cre. This was an interesting observation consistent with the expression of Pou4f2 in the male and female germline and suggests that the Pou4f2Cre transgenic mouse line could be used to understand the genetic underpinnings of varying germline development between sexes [14,15]. In crosses, we have established using floxed alleles of the target genes that the recombination rate is complete, resulting in 100% transmission of recombined alleles, which increases the efficiency of producing mice that carry mutations that would otherwise prevent or reduce breeding efficiency.

The activity of Pou4f2Cre recombination was observed in multiple populations of cells in the retina. Here we mapped patterns of recombination using two reporter lines, Ai9 and Brainbow. The Pou4f2Cre line targets RGCs during early stages of retinal development and would be useful for studying processes such as RGC axon guidance. At later time points, recombination is observed within neurons in the retinal ganglion cell layer, the inner nuclear layer, and a small number of photoreceptors. The Brainbow reporter was observed only in RGCs, ACs, and HCs, consistent with activity of the Thy1 promoter. The expression of Cre from the Pou4f2 locus differs from immunohistological studies in which RGCs are the primary cell type of the retina expressing Pou4f2Cre and related proteins [9]. The IRES sequence added to the construct is highly GC-rich, and such sequences are likely to alter local DNA methylation patterns. We speculate that this is why the expression pattern of Cre also differs from a recently reported Pou4f2 Dre/Cre line [18]. In this line, Dre-mediated recombination replaces the Pou4f2 gene with Cre. The Cre driven from the Pou4f2 locus in this transgenic mouse is observed in the entire animal, which is different compared to the expression pattern of Pou4f2 as detected in situ [18]. These results suggest that expression driven from the Pou4f2 locus is highly sensitive to small changes, such as those made in engineering the recombinant lines and could account for the expression of Cre in non-RGC populations in the retina reported in this study. Alternatively, this could reflect low expression of Pou4f2 in these cell types (i.e., ACs).

We also report that the timing of Pou4f2Cre-mediated recombination within the retina is also highly dependent upon the cell type in which the target of recombination is located. By taking advantage of the timing of this recombination pattern, investigators will have a reproducible method to target amacrine and bipolar cells after the completion of developmental processes such as developmental cell death and dendrite outgrowth to complement the use of Cre:ER lines. Sparse recombination was also observed in the neurons of the enteric nervous system, photoreceptors, and some other regions of the brain, allowing for genetic manipulations to be targeted in a small number of cells.

In conclusion, this study characterizes a transgenic Pou4f2 knock-in Cre recombinase transgenic mouse line. This line can be used to deplete RGC number, to target germline recombination, and to target recombination in retinal neurons while sparing Müller glia and most photoreceptors.


This research was supported by R01EY020857 (Fuerst). Imaging support was provided by NIH Grants P20RR016454, P30GM103324, and P20GM103408. Author Contributions: All authors contributed to laboratory work and data collection. Writing was done by ABS and PGF. Authors have no conflicting commercial interests.


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