Generation of knock-in Cre and FlpO mouse lines for precise targeting of striatal projection neurons and dopaminergic neurons

Reviewer #1 (Public review):

Summary:

I read with much attention the manuscript titled "Generation of knock-in Cre and FlpO mouse lines for precise targeting of striatal projection neurons and dopaminergic neurons" in which the authors reveal five transgenic lines to target diverse neuronal populations of the basal ganglia. In addition, the authors also provide some assessments of the functionality of the lines.

Strengths:

Knockin lines made readily available through Jackson. Lines show specific expression.

Weaknesses:

Although I have no doubt these knocking lines will be broadly used by researchers in the field, I find the scientific advances of the study and the breadth of the resource provided quite limited. This is partly because 4 of these lines have been generated by other laboratories. For instance, there are already two other Dat-FlpO lines generated (JAX#: 033673 and 035436), with one of them already characterized (PMID: 33979604). Similarly, Drd1-Cre and Adora2a-Cre have been used abundantly since they were generated over a decade ago, and a novel Drd1-FlpO line has been characterized thoroughly recently (PMID: 38965445). Indeed, some of these lines were BAC transgenic, and I agree with the authors that there is a sound rationale for generating knock-in mice; however, the authors should then demonstrate if/how their new drivers are superior. Overall, the valuable resource generated by the authors would benefit from additional quantification and validation.

Reviewer #2 (Public review):

Summary:

The authors report the generation and validation of new knock-in mouse lines enabling precise targeting of basal ganglia projection neurons and midbrain dopamine neurons. By inserting recombinase sequences at endogenous loci, they provide tools that improve on older BAC-based models, with the additional benefit that all lines are openly available through Jackson Laboratories. This work is timely, fills a longstanding gap for the community, and will support both basic circuit mapping and disease-related research.

Strengths:

The major strength of this study is the provision of new genetic resources that will be widely used by the basal ganglia and dopamine research communities. Anatomical and electrophysiological data indicate appropriate expression and preserved intrinsic properties. The Flp lines, in particular, show labeling largely confined to basal ganglia circuits, making them especially attractive for circuit-based studies. A further strength is the use of a T2A-recombinase insertion at the native gene stop codon, which preserves endogenous regulation and maintains near-physiological expression of Adora2a, Drd1a, and DAT. The availability of both Cre and Flp versions enables powerful intersectional strategies, and open distribution through Jackson Laboratories ensures broad accessibility and long-term value.

Weaknesses:

The major limitation is the discrepancy between Cre and Flp lines, with Cre generally driving broader expression than Flp. This raises concerns about anatomical fidelity that require validation at the cellular level. For the DAT-FlpO line, efficiency remains insufficiently quantified, and higher-resolution co-labeling with TH immunostaining is needed. Electrophysiological comparisons between Cre and Flp versions are also incomplete; current data suggest potential physiological differences, which warrant additional statistical testing and, at a minimum, explicit discussion in the manuscript.

Reviewer #3 (Public review):

Summary:

Using latest knock-in technology, the authors generated a set of five mouse lines with expression of recombinases in striatal projection neurons and dopaminergic neurons for public use. They rigorously characterize the expression of the recombinases by intersectional crossing with reporter lines to demonstrate that these lines are faithful, and they perform electrophysiological experiments in slices to provide evidence that the respective neurons show the expected features in these assays.

Strengths:

The characterization of the new mouse lines is exceptional, and these will be widely used by the community. The mouse lines are openly available for the community to use.

Weaknesses:

No weaknesses were identified by this Reviewer.

Author response:

We thank all three reviewers for their thoughtful and constructive evaluations of our manuscript, “Generation of knock-in Cre and FlpO mouse lines for precise targeting of striatal projection neurons and dopaminergic neurons.” We are encouraged that the reviewers recognize the value, specificity, and utility of these new lines for the basal ganglia and dopamine research communities. Below, we summarize our planned revisions and clarifications in response to the reviewers’ comments.

(1) Novelty and comparison with existing lines

We appreciate Reviewer 1’s point regarding the existence of previously generated Cre and Flp lines targeting similar neuronal populations. Our project was initiated six years ago, and during the course of generating and characterizing all five lines, we became aware that similar individual lines have since been developed by other groups. Nevertheless, our study provides a coordinated and independently validated set of lines created using a standardized knock-in (KI) strategy and distributed through Jackson Laboratories for unrestricted community use. Importantly, whereas previous BAC transgenic approaches rely on random insertion, which can lead to position effects and ectopic expression, our design places the recombinase coding sequence immediately downstream of the endogenous stop codon using a self-cleaving T2A peptide. This ensures expression under native promoter and regulatory control, preserving physiological gene regulation.

To address the Reviewers’ points, we will (i) expand the Introduction and Discussion to clarify the rationale and advantages of endogenous promoter–driven recombinase expression over BAC-based systems, emphasizing that our lines provide a uniform, promoter-controlled, and publicly accessible toolkit for the community, (ii) and explore including a comparative table summarizing differences in construct design, expression fidelity, and recombination efficiency across published lines (e.g., PMID 33979604, 38965445).

(2) Quantification, validation, and comparison of Cre vs FlpO

We agree with Reviewers 1 and 2 that further quantification and discussion of Cre versus FlpO fidelity will strengthen the manuscript. The observed difference in expression breadth between Cre and FlpO lines likely reflects a fundamental property of the recombinases themselves rather than a discrepancy in targeting. Cre recombinase is significantly more enzymatically efficient than FlpO, meaning that even very low endogenous levels of gene expression (e.g., Drd1a or Adora2a) can drive Cre-dependent recombination, whereas FlpO requires higher expression thresholds. Consequently, reporter-based readouts will inherently appear broader for Cre lines, despite both being driven by the same endogenous promoters.

To address these points, we will (i) provide quantitative co-labeling analyses for the DAT-FlpO line with TH immunostaining to assess efficiency and specificity, (ii) clarify in the Results and Discussion that differences between Cre and FlpO expression patterns largely stem from differences in recombinase kinetics and sensitivity, not mismatched promoter activity, (iii) and include representative high-resolution images and relevant statistics in the revised figures. Importantly, we would like to note that RNAscope may not be an ideal validation approach in this context, as in situ transcript detection cannot capture the enzymatic threshold differences that determine reporter recombination and thus will not help address observed differences between Cre and FlpO lines. Finally, we are actively performing electrophysiological comparisons between Cre and FlpO lines to rigorously quantify potential physiological differences between them. Updated analyses will be incorporated as available or described as ongoing future work.

(3) Discussion of scope and interpretation

We appreciate the reviewers’ suggestions to better contextualize the scope of this resource. We will revise the Discussion to (i) highlight that the Cre–FlpO pairings enable powerful intersectional and cross-line strategies for dissecting basal ganglia and midbrain circuitry, (ii) and clarify that our goal was to generate a rigorously validated foundational resource, with detailed functional comparisons and manipulation studies to be explored in subsequent work.

In summary, we thank the reviewers for their insightful feedback. The planned revisions and clarifications will underscore the unique strengths of our knock-in design, explore potential Cre–FlpO differences, and highlight the value of this standardized and accessible toolkit for the neuroscience community.