Evidence for RNA or protein transport from somatic tissues to the male reproductive tract in mouse
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eLife assessment
This manuscript reports data consistent with a new and unanticipated phenomenon: that Cre or its mRNA may be transmitted between tissues in the mouse and that the male reproductive tract (epididymis) appears to be the most common target of such transported molecules. The data serve as a timely warning to mouse researchers about an unexpected complication of Cre-mediated gene manipulation.
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Abstract
The development of tools to manipulate the mouse genome, including knockout and transgenic technology, has revolutionized our ability to explore gene function in mammals. Moreover, for genes that are expressed in multiple tissues or at multiple stages of development, the use of tissue-specific expression of the Cre recombinase allows gene function to be perturbed in specific cell types and/or at specific times. However, it is well known that putative tissue-specific promoters often drive unanticipated ‘off-target’ expression. In our efforts to explore the biology of the male reproductive tract, we unexpectedly found that expression of Cre in the central nervous system resulted in recombination in the epididymis, a tissue where sperm mature for ~1–2 weeks following the completion of testicular development. Remarkably, we not only observed reporter expression in the epididymis when Cre expression was driven from neuron-specific transgenes, but also when Cre expression in the brain was induced from an AAV vector carrying a Cre expression construct. A surprisingly wide range of Cre drivers – including six different neuronal promoters as well as the adipose-specific Adipoq Cre promoter – exhibited off-target recombination in the epididymis, with a subset of drivers also exhibiting unexpected activity in other tissues such as the reproductive accessory glands. Using a combination of parabiosis and serum transfer experiments, we find evidence supporting the hypothesis that Cre may be trafficked from its cell of origin to the epididymis through the circulatory system. Together, our findings should motivate caution when interpreting conditional alleles, and suggest the exciting possibility of inter-tissue RNA or protein trafficking in modulation of reproductive biology.
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eLife assessment
This manuscript reports data consistent with a new and unanticipated phenomenon: that Cre or its mRNA may be transmitted between tissues in the mouse and that the male reproductive tract (epididymis) appears to be the most common target of such transported molecules. The data serve as a timely warning to mouse researchers about an unexpected complication of Cre-mediated gene manipulation.
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Reviewer #1 (Public Review):
The authors report data consistent with a new and unanticipated phenomenon: that Cre or its mRNA may be transmitted between tissues in the mouse. The epididymis appears to be the most common beneficiary of such transport from neural, and some other, tissues. The authors show this in two ways. First, they infect brains with AAV expressing Cre. They see expression of TdTomato in epididymis, from a construct that cannot express unless a loxP-flanked STOP cassette is recombined out. Second, because viral spread is a possible confounding artifact of AAV delivery, the authors also show that transgenes that drive Cre expression in the nervous system or elsewhere can cause TdTomato expression in the epididymis. They rule out that TdTomato is itself transmitted to the epididymis by showing that recombination occurred …
Reviewer #1 (Public Review):
The authors report data consistent with a new and unanticipated phenomenon: that Cre or its mRNA may be transmitted between tissues in the mouse. The epididymis appears to be the most common beneficiary of such transport from neural, and some other, tissues. The authors show this in two ways. First, they infect brains with AAV expressing Cre. They see expression of TdTomato in epididymis, from a construct that cannot express unless a loxP-flanked STOP cassette is recombined out. Second, because viral spread is a possible confounding artifact of AAV delivery, the authors also show that transgenes that drive Cre expression in the nervous system or elsewhere can cause TdTomato expression in the epididymis. They rule out that TdTomato is itself transmitted to the epididymis by showing that recombination occurred in the epididymis of the TdTomato-expressing mice.
I believe that the authors saw what they report. The data are beautiful and convincing, the experimental design was excellent in every sense, including the use of multiple alternative Cre lines, viruses, or methods. The expression of TdTomato in the epididymis and sometimes elsewhere was unambiguous, and using PCRs to validate editing in TdTomato expressing cells clinched the case.
What I am less sure of is the interpretation. The creative idea that Cre or its RNA can move between tissues in mouse would be extremely important for future technical exploitation and for demonstrating a previously un-considered complication to interpreting mouse reverse-genetic results. But as the authors note, both experiments to show this have potential caveats: AAV could escape into the circulation and go to other tissues, and promoter-Cre fusions can have leaky expression outside their expected expression zone. The authors argue, appropriately, that these most likely artifacts in the two experiment types differ, so one would have to posit that both types of artifacts occur. But this is not impossible.
I was thus excited for the parabiosis experiment, as it was the perfect way to settle the issue. The choice of strains to link in this way was ideal. Unfortunately, the sample size was small and the results were mixed: 1 of 3 cases showed a result consistent with the authors' hypothesis. Further experiments involving injection of exosomes or serum were similarly suggestive but not conclusive.
The clear and convincing data are a warning to mouse researchers about an unexpected complication of Cre-mediated gene manipulation. The data presented are consistent with the most interesting model, that Cre or its RNA can be transmitted between tissues, but additional data would make this conclusion unassailable.
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Reviewer #2 (Public Review):
This report highlights the unexpected off-target presence of Cre in the mouse epididymis under conditions where specific Cre activity was only expected in the brain or adipose tissue. The use of a modified CLARITY protocol to provide visual demonstration of Cre in the caput epididymis was complemented and strengthened by supplementary data from fluorescent microscopy. However, the apparent '2-phase' expression between the distal and proximal portions of the caput was not further elaborated upon.
Through a series of technically challenging studies involving parabiosis and serum/exosome transfer experiments, there was some evidence that off-target expression involved the circulatory system. However, the lack of consistent outcomes suggests that this is not a robust effector process, so the precise reasons for …
Reviewer #2 (Public Review):
This report highlights the unexpected off-target presence of Cre in the mouse epididymis under conditions where specific Cre activity was only expected in the brain or adipose tissue. The use of a modified CLARITY protocol to provide visual demonstration of Cre in the caput epididymis was complemented and strengthened by supplementary data from fluorescent microscopy. However, the apparent '2-phase' expression between the distal and proximal portions of the caput was not further elaborated upon.
Through a series of technically challenging studies involving parabiosis and serum/exosome transfer experiments, there was some evidence that off-target expression involved the circulatory system. However, the lack of consistent outcomes suggests that this is not a robust effector process, so the precise reasons for the off-target expression remain unknown.
This study raises more questions than uncovered answers, and the conclusions are somewhat speculative (and correctly so). We are not closer to understanding why there is off-target Cre expression, nor why it is limited to the epididymis. It is not apparent how, and if, this unexpected observation holds any implications on past research reliant on Cre-recombination if those studies do not focus on the male reproductive tract, or the animal's health/behaviour is not affected. However, there is initial evidence (albeit less robust than desired) to support the authors' claim of distal organ-to-organ signalling, consistent with previous reports. Overall, this study currently speaks more so to the technology, rather than systems biology.
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Reviewer #3 (Public Review):
The Cre-Loxp leakage phenomenon in the transgenic mice have been noticed for year. The current study systematically applied multiple "tissue-specific" Cre mouse lines and found that the mouse epididymis is a hot spot for the Cre-Lopx off-target effect. The authors try to demonstrate that the off-target effect in the epididymis could be mediated by the transfer of Cre mRNA/protein molecules from the original Cre-expressing tissue (e.g. brain). Their conclusions are partially supported by the serum/exosome transfer experiment and parabiotic pair experiments (only 1 parabiotic pairs shows positive result, while others didn't).
Overall, these experiments involve lots of works and should be appreciated by the field. However, the paper didn't stringently test the other possibility of Cre-Loxp leakage phenomenon, …
Reviewer #3 (Public Review):
The Cre-Loxp leakage phenomenon in the transgenic mice have been noticed for year. The current study systematically applied multiple "tissue-specific" Cre mouse lines and found that the mouse epididymis is a hot spot for the Cre-Lopx off-target effect. The authors try to demonstrate that the off-target effect in the epididymis could be mediated by the transfer of Cre mRNA/protein molecules from the original Cre-expressing tissue (e.g. brain). Their conclusions are partially supported by the serum/exosome transfer experiment and parabiotic pair experiments (only 1 parabiotic pairs shows positive result, while others didn't).
Overall, these experiments involve lots of works and should be appreciated by the field. However, the paper didn't stringently test the other possibility of Cre-Loxp leakage phenomenon, which is due to transcriptional leakage of the Cre system in the epididymal tissue. Also, the inconsistent of result from parabiosis within limited animal replicates, the questionable quality of PCR results in multiple figures has led to an uncertainty of the conclusion.
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