Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity

No leakiness was reported in those studies using the YFP reporter line to verify two inducible Cx3cr1-CreERT2 lines.

“No leakiness was reported in those studies.”

• cites Jung et al., 2000 sourceCould not verify

  The Results and Discussion describes a CX3CR1–EGFP knock-in mouse, not YFP reporter validation of two inducible Cx3cr1-CreERT2 lines. Searches for YFP, inducible Cre and leakiness found no passage addressing whether those lines showed uninduced reporter activity. Page numbers were not available in our copy. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites Parkhurst et al., 2013 sourceDisputed

  The Results section, “Generation of CX3CR1CreER mice to manipulate gene expression in microglia” (page not available), reports DsRed-positive microglia even without tamoxifen, contrary to the claim that no leakiness was reported. EYFP marked cells expressing the CreER transcript; the paper used a DsRed reporter to test Cre-mediated recombination. (LLM, conf=0.89)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

The inducible Cx3cr1-CreERT2 line developed by Jung et al. (2000) displayed some basal cre activity without tamoxifen treatment.

“The inducible Cx3cr1-CreERT2 line developed by Jung et al. (2000), displayed some basal cre activity without tamoxifen treatment, but this expression was restricted to microglia.”

• Also stated in: § Discussion · plain

  “We found that this inducible line can drive expression of GFP reporter in a fraction of microglia before tamoxifen treatment.”

• cites Jung et al. (2000) sourceDisputed

  The Results and Discussion describe a CX3CR1–GFP knock-in mouse, not an inducible Cx3cr1-CreERT2 line. Materials and Methods say Cre was introduced transiently into embryonic stem cells to remove a selection gene; the paper does not report basal Cre activity or tamoxifen treatment. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

This basal cre activity in the Cx3cr1-CreERT2 line developed by Jung et al. (2000) was restricted to microglia.

“The inducible Cx3cr1-CreERT2 line developed by Jung et al. (2000), displayed some basal cre activity without tamoxifen treatment, but this expression was restricted to microglia.”

• cites Jung et al. (2000) sourceDisputed

  The Results and Discussion describe a CX3CR1-GFP reporter mouse, not an inducible Cx3cr1-CreERT2 line. Materials and Methods say Cre was used transiently in embryonic stem cells to remove a selection cassette; the paper does not report basal Cre activity or tamoxifen treatment. Its GFP expression was also observed in peripheral blood monocytes, not only microglia. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

According to The Jackson Laboratory website, the constitutive Cx3cr1-Cre line created by Jung et al. (2000) displays leakiness into neurons when crossed with a yellow fluorescent protein (YFP) reporter line.

“According to the description on The Jackson Laboratory website, the constitutive Cx3cr1-Cre line created by Jung et al. (2000), displays leakiness into neurons when crossed with a yellow fluorescent protein (YFP) reporter line.”

• cites Jung et al. (2000) sourceDisputed

  The Results and Discussion section (‘Generation of CX3CR-1GFP mice’; page not provided) says Jung et al. generated a CX3CR1–EGFP replacement line, not a constitutive Cx3cr1-Cre line. The Methods say Cre was used transiently in embryonic stem cells to remove the neo gene. The paper does not report a YFP-reporter cross or neuronal leakiness. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

The partial tamoxifen-independent cre activity observed in the Cx3cr1-CreERT2 line developed by Jung et al. (2000) indicates potential embryonic deletion of TSC1 in a fraction of microglia.

“The partial tamoxifen-independent cre activity observed in the Cx3cr1-CreERT2 line developed by Jung et al. (2000), indicates potential embryonic deletion of TSC1 in a fraction”

• cites Jung et al. (2000) sourceDisputed

  The Results and Discussion section identifies the line Jung et al. generated as CX3CR1-GFP mice, not Cx3cr1-CreERT2 mice. Materials and Methods says Cre was used transiently in embryonic stem cells to remove a selectable marker; it does not report tamoxifen-independent Cre activity or TSC1 deletion in microglia. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

The cre activity in the Cx3cr1-CreERT2 line developed by Jung et al. (2000) is not fully dependent on tamoxifen.

“Thus, the cre activity in this line is not fully dependent on tamoxifen.”

• cites Jung et al. (2000) sourceDisputed

  The Results and Discussion (page not available) identify the line Jung et al. generated as CX3CR1-GFP, with EGFP replacing part of the CX3CR1 gene—not a Cx3cr1-CreERT2 line. Materials and Methods (page not available) mention Cre only as a vector transiently introduced into embryonic stem cells to remove a selection gene. The paper does not assess tamoxifen dependence. (LLM, conf=0.86)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

The inducible Cx3cr1-CreERT2 mouse line developed by Parkhurst et al. displayed a reasonable level of microglial-specificity in their hands.

“The inducible Cx3cr1-CreERT2 mouse line that was developed by Parkhurst et al., displayed a reasonable level of microglial-specificity in their hands (Parkhurst et al., 2013).”

• cites Parkhurst et al., 2013 sourceSupported

This cre-independent leakage likely accounts, at least in part, for the GFP-positive non-microglial cells observed in Zhang et al. (2018).

“This cre-independent leakage likely accounts, at least in part, for the GFP-positive non-microglial cells observed in that previous study (Zhang et al., 2018).”

• cites Zhang et al., 2018 sourceSupported

Our study suggests the Rosa-Greenf/f reporter line itself could contribute to the leakage into neurons and other cells noted by others (Zhang et al., 2018).

“Our study suggests that the Rosa-Greenf/f reporter line itself could contribute to the leakage into neurons and other cells that was noted by others (Zhang et al., 2018).”

• cites Zhang et al., 2018 sourceSupported

Zhang et al. (2018) reported that the Parkhurst Cx3cr1-CreERT2 line has significant leakage into neurons.

“However, Zhang et al. (2018) reported that this line has significant leakage into neurons.”

• cites Zhang et al., 2018 sourcePartly supported

  The Results (page unavailable) report neuronal Cre-recombination, but the large neuronal involvement was found with constitutive Cx3cr1-Cre. For the inducible Cx3cr1-CreER line, they report about 5% of cortical neurons showing recombination; Materials and Methods (page unavailable) identify that line as Litt/Wgan, not Parkhurst. The paper therefore does not establish significant neuronal leakage in the Parkhurst line. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

The Rosa-Greenf/f reporter line used in a previous study by Zhang et al. (2018) shows spontaneous leakiness into non-microglial cells in the absence of cre recombinase.

“In the present study, we unexpectedly found that the Rosa-Greenf/f reporter line used in a previous study by other researchers (Zhang et al., 2018) shows spontaneous leakiness into non-microglial cells in the absence of cre recombinase.”

• Also stated in: § Discussion · plain

  “Surprisingly, however, the Rosa-Greenf/f and tdTomatof/f lines displayed spontaneous leakiness independent of cre recombinase.”

• cites Zhang et al., 2018 sourceSupported

The YFP reporter line was used to verify two inducible Cx3cr1-CreERT2 lines (Jung et al., 2000; Parkhurst et al., 2013).

“The YFP reporter line was used to verify two inducible Cx3cr1-CreERT2 lines (Jung et al., 2000; Parkhurst et al., 2013).”

• cites Jung et al., 2000 sourcePartly supported

  The Results and Discussion section (page not provided) describes a Cx3cr1 EGFP reporter mouse line, supporting only the identification of a Cx3cr1 fluorescent reporter. It describes neither a YFP reporter nor the use of that line to verify two inducible Cx3cr1-CreERT2 lines. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites Parkhurst et al., 2013 sourcePartly supported

  The Results section, “Generation of CX3CR1CreER mice” (page not provided), describes one inducible line with a built-in EYFP element and uses EYFP to assess its expression. It does not say that a separate YFP reporter line verified two inducible lines; Cre activity was tested with a DsRed reporter instead. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Among the three fluorescent reporters tested, the mT/mG reporter line appears to consistently deliver GFP reporter expression in a cre-dependent manner.

“Among the three fluorescent reporters tested, the mT/mG reporter line appears to consistently deliver GFP reporter expression in a cre-dependent manner, not only in the present study, but also in many other studies (Zhao et al., 2018).”

• cites Zhao et al., 2018 sourcePartly supported

  The Experimental Procedures (Animals; page unavailable) identify the Rosa mT/mG line among the mice used, so Zhao et al. is an example of a study using that line. Our copy does not report whether the line consistently produced Cre-dependent GFP expression or compare it with other fluorescent reporters. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

No spontaneous seizures were observed in the Zhang et al. (2018) study that used the Parkhurst Cx3cr1-CreERT2 line.

“In contrast, no spontaneous seizures were observed in the other study (Zhang et al., 2018) that used the Cx3cr1-CreERT2 mouse line developed by Parkhurst et al. (2013).”

• cites Zhang et al., 2018 sourceSupported
• cites Parkhurst et al. (2013) sourceSupported

On tamoxifen treatment, a fraction of mice in Zhao et al. (2018) displayed spontaneous seizures.

“On tamoxifen treatment, a fraction of mice displayed spontaneous seizures.”

• cites Zhao et al. (2018) sourceSupported

This potential embryonic deletion of TSC1 may explain the differences in seizure susceptibility observed in these two studies (Jung et al., 2000; Zhang et al., 2018; Zhao et al., 2018).

“which may explain the differences in seizure susceptibility observed in these two studies (Jung et al., 2000; Zhang et al., 2018; Zhao et al., 2018).”

• cites Jung et al., 2000 sourceCould not verify

  The Results and Discussion describe CX3CR1 deletion and GFP expression in microglia, while Materials and Methods describe the targeting procedure. Neither section addresses TSC1 deletion, seizure susceptibility, or differences between the two studies; searches for those terms found no supporting passage in our copy. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites Zhang et al., 2018 sourcePartly supported

  The Discussion says constitutive Tsc1 deletion involved presumed embryonic induction and that prenatal, unlike postnatal, microglial deletion might be sufficient to cause epilepsy. But it does not propose embryonic deletion as the explanation for the seizure difference between the two studies: it notes that the other study also found seizures after postnatal deletion and discusses neuronal involvement, genetic background, and recombination efficiency as possible factors. Page numbers are unavailable in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites Zhao et al., 2018 sourcePartly supported

  The Results section (page unavailable) reports seizures after TSC1 deletion in microglia and acknowledges that deletion during brain development could contribute to epilepsy. It also reports seizures after deletion in adult mice. The paper does not attribute differences in seizure susceptibility between the two cited studies to potential embryonic deletion. (LLM, conf=0.80)

  Experimental — accuracy not yet measured

In Zhao et al. (2018), the Cx3cr1-CreERT2 line developed by Jung et al. (2000) was employed to deplete TSC1 in microglia.

“In the study by Zhao et al. (2018), this line was employed to deplete TSC1 in microglia.”

• cites Zhao et al. (2018) sourcePartly supported

  The Results section (‘Elevated mTOR Signaling in Microglia Is Sufficient to Promote the Development of SRSs’; page unavailable) confirms that Zhao et al. crossed a Cx3cr1-CreERT2 line with TSC1-floxed mice to delete TSC1 in microglia. It identifies the Cx3cr1-CreERT2 line with Parkhurst et al. (2013), not Jung et al. (2000), so the claim's identification of the line is unsupported. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

A floxed fluorescent reporter mouse line frequently used to verify Cre lines displays spontaneous GFP reporter expression independent of Cre recombinase.

“Here, we found that a floxed fluorescent reporter mouse line which has been frequently used to verify Cre lines displayed spontaneous expression of the GFP reporter, independent of Cre recombinase, thus revealing a potential caveat in assessing cre lines.”

In Rosa-Greenf/f mice examined at around two to six months of age, 33.33% of animals displayed significant GFP reporter expression throughout the brain, from sporadic cells to clusters.

“To our surprise, 33.33% of the animals displayed significant expression of GFP reporter throughout the brain, from individually sporadic GFP-positive cells to clusters (Fig. 1A,C; Extended Data Fig. 1-1A).”

In vehicle treated Cx3cr1-CreERT2J±;mT/mGf/- mice, up to 18.9 ± 4.3% of Iba1+ cells are GFP positive in the cortex.

“In vehicle treated mice, up to 18.9 ± 4.3% and 23.0 ± 3.4% of Iba1+ cells are GFP positive in the cortex and hippocampus, respectively.”

Occasionally, a small fraction of tdTomato mice displayed very broad spontaneous leakage.

“Of note, occasionally, a small fraction of tdTomato mice displayed very broad spontaneous leakage.”

A significant portion of GFP-positive cells in Rosa-Greenf/f mice are Iba1/NeuN/GFAP triple negative: 79.8% in cortex and 57.8% in hippocampus.

“Therefore, a significant portion of GFP-positive cells are Iba1/NeuN/GFAP- triple negative in the cortex (79.8%) and hippocampus (57.8%; two males and one female).”

The Cx3cr1-CreM line from MMRRC is reasonably microglia specific.

“We have also confirmed that the Cx3cr1-CreM line from MMRRC is reasonably microglia specific.”

• Also stated in: § Discussion · plain

  “In the present study, we verified that the constitutive Cx3cr1-CreM line from the MMRRC is microglia specific.”

In Cx3cr1-Cre±;mT/mGf/- mice after SE, few, if any, NeuN+/GFP+ double-positive cells were seen.

“We saw few, if any, NeuN+/GFP+ or GFAP+/GFP+ double-positive cells.”

A GFP reporter mouse line used in a previous study displays spontaneous leakiness into neurons, independent of Cre recombinase.

“We found that a GFP reporter mouse lines used in a previous study displays spontaneous leakiness into neurons, independent of Cre recombinase.”

The tdTomatof/f line displayed spontaneous leakiness independent of cre recombinase.

“Surprisingly, however, the Rosa-Greenf/f and tdTomatof/f lines displayed spontaneous leakiness independent of cre recombinase.”

None of the other GFP+ cells are GFAP-positive in Cx3cr1-CreM±;Rosa-Greenf/- mice after SE.

“Among the other GFP+ cells, only 0.31 ± 0.54% and 0.07 ± 0.13% are NeuN positive in the cortex and hippocampus, respectively and none of them are GFAP-positive (Fig. 4A–D).”

In the hippocampus of Rosa-Greenf/f mice, Iba1+, NeuN+, and GFAP+ populations among GFP-positive cells are 0%, 41.71 ± 15.45%, and 0.47 ± 0.83%, respectively.

“In the hippocampus, Iba1+, NeuN+, and GFAP+ populations are 0%, 41.71 ± 15.45%, and 0.47 ± 0.83%, respectively.”

In Cx3cr1-CreM±;Rosa-Greenf/- mice after pilocarpine-induced SE, 98.8 ± 1.1% of GFP+ cells were Iba1-positive in the cortex.

“In Cx3cr1-CreM±;Rosa-Greenf/-, we found that 98.8 ± 1.1% and 99.6 ± 0.24% of GFP+ cells were Iba1-positive in the cortex and hippocampus, respectively (two males and one female).”

In Cx3cr1-Cre±;mT/mGf/- mice after SE, few, if any, GFAP+/GFP+ double-positive cells were seen.

“We saw few, if any, NeuN+/GFP+ or GFAP+/GFP+ double-positive cells.”

In Cx3cr1-Cre±;mT/mGf/- mice after SE, 99.9 ± 0.13% of GFP-positive cells were microglia in the cortex.

“Moreover, in Cx3cr1-Cre±;mT/mGf/- mice, we observed that nearly all GFP-positive cells were microglia (99.9 ± 0.13% in the cortex and 100% in the hippocampus; two males and one female; Fig. 4E–H).”

Several dozens of non-microglial GFP-positive cells were scattered sporadically throughout the entire brain in Cx3cr1-CreM±;Rosa-Greenf/- mice.

“However, we also observed several dozens of non-microglial GFP-positive cells scattered sporadically throughout the entire brain (Fig. 2A–D; Extended Data Fig. 2-1A–C).”

• AssumptionRests on an assumption: Endogenous Cx3cr1 is a selective marker for microglia in the CNS.

These two Cx3cr1-Cre mouse lines maintain microglia-specific expression even following excitatory injury.

“Finally, we clarified that these two mouse lines maintain microglia-specific expression even following excitatory injury.”

• AssumptionRests on an assumption: Endogenous Cx3cr1 is a selective marker for microglia in the CNS.

In Cx3cr1-Cre±;mT/mGf/- mice after SE, 100% of GFP-positive cells were microglia in the hippocampus.

“Moreover, in Cx3cr1-Cre±;mT/mGf/- mice, we observed that nearly all GFP-positive cells were microglia (99.9 ± 0.13% in the cortex and 100% in the hippocampus; two males and one female; Fig. 4E–H).”

All seven tdTomato reporter mice examined displayed significant leakage of tdTomato in the brain.

“We found that the seven animals examined display significant leakage of tdTomato in the brain (four males and three females; Fig. 1B,D).”

Two Cx3cr1-Cre mouse lines can drive fluorescent reporter expression largely restrictively in microglia (are microglia-specific).

“We further confirmed that two Cx3cr1-Cre mouse lines can drive fluorescent reporter expression largely restrictively in microglia.”

• Also stated in: § Significance Statement · boosted

  “Furthermore, we confirmed that two Cre-lines are microglia-specific and thus can be redeployed without hesitation.”

• AssumptionRests on an assumption: Endogenous Cx3cr1 is a selective marker for microglia in the CNS.

In Cx3cr1-Cre±;mT/mGf/- mice, 99.9 ± 0.17% and 99.7 ± 0.36% of GFP-positive cells were Iba1 positive in the cortex and hippocampus, respectively.

“We observed that among the GFP-positive cells, 99.9 ± 0.17% and 99.7 ± 0.36% were Iba1 positive in the cortex and hippocampus, respectively (Fig. 2E,G);”

• AssumptionRests on an assumption: Endogenous Cx3cr1 is a selective marker for microglia in the CNS.

In vehicle treated Cx3cr1-CreERT2J±;mT/mGf/- mice, up to 23.0 ± 3.4% of Iba1+ cells are GFP positive in the hippocampus.

“In vehicle treated mice, up to 18.9 ± 4.3% and 23.0 ± 3.4% of Iba1+ cells are GFP positive in the cortex and hippocampus, respectively.”

In Cx3cr1-CreM±;Rosa-Greenf/- mice after pilocarpine-induced SE, 99.6 ± 0.24% of GFP+ cells were Iba1-positive in the hippocampus.

“In Cx3cr1-CreM±;Rosa-Greenf/-, we found that 98.8 ± 1.1% and 99.6 ± 0.24% of GFP+ cells were Iba1-positive in the cortex and hippocampus, respectively (two males and one female).”

Lectin is colocalized with mTomato, confirming that mTomato is highly expressed in the blood vessels.

“We saw that lectin is colocalized with mTomato, confirming that mTomato is highly expressed in the blood vessels.”

• AssumptionThe authors state this as an assumption: Mice are expected to express membrane tagged-Tomato (mTomato) ubiquitously independent of Cre recombinase, whereas expression of membrane tagged-GFP (mGFP) requires Cre-mediated homologous recombination.

Among the other GFP+ cells in Cx3cr1-CreM±;Rosa-Greenf/- mice after SE, 0.31 ± 0.54% are NeuN positive in the cortex.

“Among the other GFP+ cells, only 0.31 ± 0.54% and 0.07 ± 0.13% are NeuN positive in the cortex and hippocampus, respectively and none of them are GFAP-positive (Fig. 4A–D).”

In Cx3cr1-CreM±;Rosa-Greenf/- mice, 99.2 ± 0.3% and 99.8 ± 0.16% of microglia are GFP-positive in the cortex and hippocampus, respectively.

“We found that 99.2 ± 0.3% and 99.8 ± 0.16% of microglia are GFP-positive in the cortex and hippocampus, respectively (two males and three females; Fig. 2A–D).”

• AssumptionRests on an assumption: Endogenous Cx3cr1 is a selective marker for microglia in the CNS.

None of the GFP-positive cells in Cx3cr1-Cre±;mT/mGf/- mice are either NeuN-positive or GFAP-positive.

“none of GFP+ cells are either NeuN-positive or GFAP-positive (two males and two females; Fig. 2F,H–J).”

• AssumptionRests on an assumption: Endogenous Cx3cr1 is a selective marker for microglia in the CNS.

After tamoxifen treatment, all Iba1+ cells are GFP positive in Cx3cr1-CreERT2J±;mT/mGf/- mice.

“After tomaxifen treatment, all Iba1+ cells are GFP positive (two males and one female).”

In the tdTomato mouse line, cre recombinase-independent leakage was observed predominantly in the hippocampus.

“In the tdTomato mouse line, we observed cre recombinase-independent leakage predominantly in the hippocampus, which is consistent with what is described at The Jackson Laboratory website, that the donating investigator reported a low level of fluorescent activity in this line.”

Cre-dependent expression in the Cx3cr1-CreERT2J line is nearly completely restricted to microglia.

“However, the Cre-dependent expression is nearly completely restricted to microglia (Fig. 3A–F).”

None of the GFP positive cells are GFAP-positive in Cx3cr1-CreERT2J±;mT/mGf/- mice.

“Again, none of GFP positive cells are either NeuN- or GFAP- positive.”

tdTomato signal is co-localized with lectin, particularly in the hippocampus.

“We observed that tdTomato signal is co-localized with lectin, particularly in the hippocampus.”

Some weak NeuN-positive cells in Rosa-Greenf/f mice lacked typical neuronal morphology.

“Notably, some weak NeuN-positive cells lacked typical neuronal morphology (Fig. 1C; Extended Data Fig. 2-1B,C).”

No leakage of mGFP expression was found over the entirety of the sections from all seven mT/mGf/f mice examined.

“We found no leakage of mGFP expression over the entirety of the sections from all seven mT/mGf/f mice we examined (three males and four females; Fig. 1E,F; Extended Data Fig. 1-1B).”

• AssumptionThe authors state this as an assumption: Mice are expected to express membrane tagged-Tomato (mTomato) ubiquitously independent of Cre recombinase, whereas expression of membrane tagged-GFP (mGFP) requires Cre-mediated homologous recombination.

We did not find any significant transient Cx3cr1 activity in neurons and astrocytes under the conditions that animals experienced excitatory injury.

“Finally, we did not find any significant transient Cx3cr1 activity in neurons and astrocytes under the conditions that animals have experienced excitatory injury.”

None of the GFP positive cells are NeuN-positive in Cx3cr1-CreERT2J±;mT/mGf/- mice.

“Again, none of GFP positive cells are either NeuN- or GFAP- positive.”

Among the other GFP+ cells in Cx3cr1-CreM±;Rosa-Greenf/- mice after SE, 0.07 ± 0.13% are NeuN positive in the hippocampus.

“Among the other GFP+ cells, only 0.31 ± 0.54% and 0.07 ± 0.13% are NeuN positive in the cortex and hippocampus, respectively and none of them are GFAP-positive (Fig. 4A–D).”

Cx3cr1-CreERT2J±;mT/mGf/- mice displayed significant expression of GFP reporter in microglia even without tamoxifen treatment.

“We found that Cx3cr1-CreERT2J±;mT/mGf/- mice displayed significant expression of GFP reporter in microglia even without tamoxifen treatment (one male and two females; Fig. 3A–F).”

Two previously created Cx3cr1-Cre lines remain invaluable tools for studying microglia.

“Together, our findings confirm that two previously created Cx3cr1-Cre lines remain as invaluable tools for studying microglia.”

• AssumptionRests on an assumption: Endogenous Cx3cr1 is a selective marker for microglia in the CNS.

In the Cx3cr1-CreERT2J mouse line, Cre-ERT2 recombinase possesses some basal enzymatic activity independent of tamoxifen.

“These data suggest that in this Cx3cr1-CreERT2J mouse line, Cre-ERT2 recombinase possesses some basal enzymatic activity independent of tamoxifen.”

The non-microglial GFP-positive cells in Cx3cr1-CreM±;Rosa-Greenf/- mice likely result from leakage of the Rosa-Green reporter itself.

“These non-microglial GFP-positive cells likely result from leakage of the Rosa-Green reporter itself.”

There is significant leakage of tdTomato expression into the blood vessels in the CNS in the absence of Cre recombinase.

“This data suggests that there is a significant leakage of tdTomato expression into the blood vessels in the CNS in the absence of cre recombinase.”

Those non-microglial GFP-positive cells are presumably from the spontaneous leakiness of the Rosa-Greenf/f reporter.

“Those non-microglial GFP-positive cells are presumably from the spontaneous leakiness of the Rosa-Greenf/f reporter.”

Both pairs of anti-Iba1 and anti-NeuN antibodies used to detect microglia and neurons are cell-type specific.

“Both pairs of antibodies are cell-type specific (Extended Data Fig. 1-3A,B ).”

The mT/mGf/f line is a reliable reporter for evaluating Cre lines in the CNS.

“Therefore, we conclude that the mT/mGf/f line is a reliable reporter for evaluating Cre lines in the CNS.”

• AssumptionThe authors state this as an assumption: Mice are expected to express membrane tagged-Tomato (mTomato) ubiquitously independent of Cre recombinase, whereas expression of membrane tagged-GFP (mGFP) requires Cre-mediated homologous recombination.

The Cx3cr1-CreM± line drives microglial-specific expression of floxed GFP reporters.

“Taken together, these data suggest that the Cx3cr1-CreM± line drives microglial-specific expression of floxed GFP reporters.”

• AssumptionRests on an assumption: Endogenous Cx3cr1 is a selective marker for microglia in the CNS.

Given that the Rosa-Greenf/f reporter line itself has spontaneous leakiness, this complicates the data interpretation.

“Given that the Rosa- Greenf/f reporter line itself has spontaneous leakiness, this complicates the data interpretation.”

tdTomato predominantly labels vascular structures in the CNS.

“It appears that tdTomato predominantly labels vascular structures in the CNS.”

Our data confirm there is little or no transient Cx3cr1-driven Cre activity in neurons or astrocytes in brains that experienced SE.

“Thus, our data confirm there is little or no transient Cx3cr1-driven Cre activity in neurons or astrocytes in brains that experienced SE.”

Our data with reporter lines suggest there is very minimal, if any, transient Cx3cr1 activity in neurons or astrocytes following excitatory injury.

“In contrast, our data with reporter lines suggest there is very minimal, if any, transient Cx3cr1 activity in neurons or astrocytes following excitatory injury.”

Increased gene dosage from BAC duplication may create issues including genetic instability and ectopic gene expression.

“This may create issues related to increased gene dosage, which could have unexpected effects, including genetic instability and ectopic gene expression.”

The BAC strategy may cause duplication of the genes brought in within the BAC constructs.

“A drawback of this approach is it may cause duplication of the genes brought in within the BAC constructs.”

All four microglia-targeting Cre lines were created using a BAC strategy.

“All four microglia-targeting Cre lines were created using a BAC strategy, which involves insertion of a large piece of DNA containing not only the gene promoter of interest, but also many other regulatory elements that help to yield the expected expression pattern of the endogenous gene (Gong et al., 2003, 2007).”

• cites Gong et al., 2003, 2007Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.