To assess whether the EGFP-reporter faithfully reflects the expression of the native Supv3L1 gene, Western blotting was performed on protein extracts from a variety of tissues of wild-type and Supv3L1tm6Jkl/+
animals using both anti-mSupv3L1 and anti-EGFP antibodies (). Expression was evident in all tissues tested with both antibodies, and the highest levels of Supv3L1 and EGFP proteins were detected in the testes, spleen, thymus and muscle. Somewhat lower expression was observed in the liver. Since the relative levels of the Supv3L1 and EGFP proteins were very similar, the EGFP-reporter reflects closely the expression of a native gene. Consistent with our prior studies (Paul et al. 2009
) the presence of the Neo
cassette in the targeted locus appears to have no affect on Supv3L1 promoter activity.
Figure 2 Expression levels of Supv3L1 and EGFP reporter as assayed by Western blotting. (A) Tissue extracts were prepared from 10 weeks old wild-type mouse and analyzed using rabbit antibody against mouse Supv3L1. (B) Quantification of the data shown in (A) after (more ...)
Taking advantage of this EGFP reporter, paternally inherited Supv3L1 gene expression was assessed at different stages of development. In timed mating experiments, heterozygous males (Supv3L1tm6Jkl/+) were crossed with wild-type females and fertilized eggs were isolated at E0.5 (). Among 54 one-cell zygotes examined, none were found to express EGFP (). Two-cell embryos isolated from oviducts at E1.5 (or obtained by overnight culture of fertilized eggs) showed no EGFP fluorescence signal (). Thus, the reporter gene is silent at these stages of development, or its expression is below the sensitivity of this detection method. First signs of expression were seen at the blastocyst stage (E3.5). Although the signal was weak, approximately 50% of blastocysts were found to display EGFP fluorescence above background (). The signal was detectable both within the inner cell mass and the trophectoderm. Blastocyst outgrowths in culture showed expression primarily in inner cell mass derived cells, but weak fluorescence was also detectable in the surrounding cells of the trophoblast lineage (not shown). Consistently, targeted ES cell clones used to derive the Supv3L1tm6Jkl/+ F1 mice were found positive for EGFP-reporter expression (not shown).
Figure 3 The EGFP reporter expression at preimplantation stages. (A, A’) One-cell embryos derived from matings of Supv3L1tm6Jkl/+ males and wild type females (paternal transmission of the reporter allele). Note no detectable expression of the EGFP transgene. (more ...)
One-cell embryos (E0.5) obtained from matings of Supv3L1tm6Jkl/+ females and wild-type males, in which the EGFP reporter is maternally transmitted, were uniformly EGFP positive (), and high level expression continued at the two-cell stage (E1.5; ). Since half of these embryos do not carry the EGFP knock-in allele, the EGFP reporter protein must have accumulated during pre-meiotic or meiotic divisions of oogenesis at which cells of this lineage existed at 2N stage. High levels of EGFP persisted in the zygotic cytoplasm until E1.5. At the blastocyst stage (E3.5), approximately half of these embryos were positive for EGFP, showing low levels of expression similar to the paternally inherited allele (). In this context, it might be important to note that meiotic reduction of 2N chromosomes to N chromosomes in Supv3L1tm6Jkl/+ mice does not affect the viability of gametes. Matings of Supv3L1tm6Jkl/+ females with wild-type males as wells as matings of Supv3L1tm6Jkl/+ males with wild-type females both produced Supv3L1tm6Jkl/+ and wild-type pups at the expected 1:1 Mendelian ratios.
At later developmental stages, the expression patterns of the paternally and maternally transmitted reporter alleles were indistinguishable (not shown). Paternally inherited expression at the late primitive streak stage (E7.5) is shown in . The expression is strong in all cell layers, including extra-embryonic tissues such as the visceral yolk sac. As expected, extra-embryonic tissues of maternal origin that do not carry the transgene, including spongy layers of endometrial tissue, appeared negative. Widespread expression of the reporter continued through the stage of 21–29 somites (E9.5; ), 43–48 pairs of somites (E11.5; ), and 60 pairs of somites (E13.5; ). The liver at E13.5 displayed detectable but lower fluorescence (). At E16.5 and at birth, all tissues examined expressed the reporter (), including extraembryonic membranes such as yolk sac () and amnion (not shown).
Figure 4 The EGFP reporter expression in postimplantation embryos. (A) Supv3L1tm6Jkl/+ embryo at E7.5 (+/−) and wild-type embryo (wt) photographed in the bright field. (B) EGFP emission of the same embryos shown in (A). (C) Merged images of (A) and (B). (more ...)
Supv3L1 expression after birth was assessed by dissecting out a variety of organs from 5 days old pups and examined for EGFP fluorescence emission. The EGFP reporter continued to be expressed widely and was detectable in all organs examined. Relatively low fluorescence was observed in the liver. The strongest expression of the reporter at 5 days of age was found in the skin, skeletal muscle, heart, brain, and eyes (not shown).
This widespread expression continued into adult life. At 10 weeks of age EGFP emission was detectable in all tissues tested (), although the levels of fluorescence appeared somewhat diminished in most of the organs relative to the expression seen at 5 days. The lowest signals were seen in the liver, lung and skin, while the highest signals were found in the testes, brain, eyes and thymus. The testes displayed the highest levels of EGFP fluorescence light emission, suggesting an important role for Supv3L1 in the spermatogenetic proliferation of cells taking place within the seminiferous tubules. The bright fluorescent eyes could actually be used for visual genotyping of both the mature transgenic and knock-in mice using an external 488 nm excitation source and EGFP-goggles. Expression of the EGFP reporter in coronal cross-sections of the brain was widespread. However, the hippocampal formations, thalamus, hypothalamus and amygdala were noticeably more fluorescent relative to the neocortices (not shown).
Figure 5 (A) Expression of EGFP reporter in tissue dissected out from 10 weeks old wild-type (wt) and Supv3L1tm6Jkl/+ (+/−) male. Images were taken at the same conditions of magnification, brightness, gain and exposure time, to reflect relative intensities (more ...)
While the EGFP reporter in the knock-in animals was integrated precisely into the Supv3L1 locus and is expressed from the native promoter, in the transgenic lines the genomic location and copy number of the transgene are not known. In these animals the Supv3L1 promoter sequence driving EGFP reporter expression is limited to 6.5 kb. Thus, it was of interest to compare the EGFP reporter expression patterns in these two lines. Similar to the knock-in mice, we found that the expression of the paternally transmitted reporter in the transgenic strain begins early during development. One-cell fertilized eggs and two-cell embryos did not express the reporter. The EGFP emission was detectable in blastocysts and became widespread at later stages (not shown). With the exception of the testes, however, the intensity of reporter expression in many tissues isolated from mature transgenic mice was found to be lower (). In fact, lung, spleen, kidney and liver from 10 weeks old transgenic animals showed EGFP emission close to the limit of detection. The reasons for the lower levels of EGFP reporter expression in adult transgenic mice are not known at this time. Although the copy number of the transgene has not been determined, pronuclear injection of DNA typically results in the tandem integration of multiple copies. Thus, the copy number of the reporter is likely to be higher in the transgenic mice relative to the single copy in knock-in animals. The factors affecting the expression EGFP levels in the transgenic mice may include position effects at the genomic locus of transgene integration (which is currently unknown), or promoter functions that may be missing in the 6.5 kb sequence used to drive reporter expression.
Finally, we tested whether the levels of reporter expression in the knock-in animals were sufficient to detect clear fluorescence signals in thin (5 microns) tissue cross-sections. The eye was chosen as an example of a highly fluorescent tissue and skin as an example of a tissue with lover levels of the reporter expression. demonstrate that EGFP fluorescence is detectable in all layers of the skin relative to the wild-type skin (). In the retina, expression of EGFP was detected predominantly in the photoreceptor layer. As shown in , the EGFP was observed in the photoreceptor inner segment and the synaptic (outer plexiform) layer. Photoreceptors are highly metabolically active neurons as they undergo periodic outer segment disc shedding and renewal (Young 1976
). Hence, photoreceptors consume large amounts of ATP generated by oxidative phosphorylation in the mitochondrial electron transport chain (Yu et al. 2001
; Yu et al. 2005
). The fact that mitochondria are predominantly located in the photoreceptor inner segment (Hoang et al 2002
; Perkins et al. 2003
) is consistent with an important role of Supv3L1 in enabling high mitochondrial activity during light-dependent signal transduction cascades.
Fig. 6 Detecting EGFP emission in tissue sections. (A) H&E stained section of the skin derived from Supv3L1tm6Jkl/+ animal. (B) Fluorescent light emission from the same section before H&E staining. Note that all cell layers including subcutaneous (more ...)
In summary, we have generated both transgenic and a knock-in mouse strains that express an EGFP reporter under control of the Supv3L1 promoter. We found that paternally inherited reporter expression begins early in development, being first detectable at the blastocyst stage. Abundant and widespread expression persists during all developmental stages as well as during postnatal growth. Expression diminishes somewhat in most adult tissues, but it remains high in testes, brain, and certain types of sensory organs and cells, such as the retina. The spatial and temporal patterns of expression were indistinguishable in the transgenic and knock-in animals. Expression levels were more faithfully maintained during adulthood in the knock-in mice and this line is likely to produce more reliable results in future experiments. However, both the transgenic and knock-in strains constitute valuable research tools that will facilitate future non-invasive studies of the intriguing biology of Supv3L1 in whole animals, as well as in ex vivo cell culture models derived from them.