Systematic screening for skin, hair, and nail abnormalities in a large-scale knockout mouse program

John P Sundberg; Soheil S Dadras; Kathleen A Silva; Victoria E Kennedy; Gaven Garland; Stephen A Murray; Beth A Sundberg; Paul N Schofield; C Herbert Pratt

doi:10.1371/journal.pone.0180682

. 2017 Jul 10;12(7):e0180682. doi: 10.1371/journal.pone.0180682

Systematic screening for skin, hair, and nail abnormalities in a large-scale knockout mouse program

John P Sundberg ^1,^*, Soheil S Dadras ², Kathleen A Silva ¹, Victoria E Kennedy ¹, Gaven Garland ¹, Stephen A Murray ¹, Beth A Sundberg ¹, Paul N Schofield ^1,³, C Herbert Pratt ¹

Editor: Marlon R Schneider⁴

¹The Jackson Laboratory, Bar Harbor, Maine, United States of America

²Departments of Dermatology and Pathology, University of Connecticut, Farmington, Connecticut, United States of America

³Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

⁴Ludwig-Maximilians-Universitat Munchen, GERMANY

Competing Interests: JPS, KAS, and VEK have a research contract with BIOCON LLC which has no relevance to this project. The remaining authors (SSD, SAM, BAS, PNS, and CHP) have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

^✉

* E-mail: john.sundberg@jax.org

Roles

John P Sundberg: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Soheil S Dadras: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Writing – original draft, Writing – review & editing

Kathleen A Silva: Data curation, Investigation, Resources, Visualization, Writing – review & editing

Victoria E Kennedy: Data curation, Investigation, Resources, Writing – review & editing

Gaven Garland: Data curation, Investigation, Resources, Writing – review & editing

Stephen A Murray: Conceptualization, Funding acquisition, Methodology, Resources, Writing – original draft, Writing – review & editing

Beth A Sundberg: Investigation, Resources, Software, Visualization, Writing – review & editing

Paul N Schofield: Conceptualization, Formal analysis, Validation, Visualization, Writing – original draft, Writing – review & editing

C Herbert Pratt: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Marlon R Schneider: Editor

PMCID: PMC5503261 PMID: 28700664

Abstract

The International Knockout Mouse Consortium was formed in 2007 to inactivate (“knockout”) all protein-coding genes in the mouse genome in embryonic stem cells. Production and characterization of these mice, now underway, has generated and phenotyped 3,100 strains with knockout alleles. Skin and adnexa diseases are best defined at the gross clinical level and by histopathology. Representative retired breeders had skin collected from the back, abdomen, eyelids, muzzle, ears, tail, and lower limbs including the nails. To date, 169 novel mutant lines were reviewed and of these, only one was found to have a relatively minor sebaceous gland abnormality associated with follicular dystrophy. The B6N(Cg)-Far2^{tm2b(KOMP)Wtsi}/2J strain, had lesions affecting sebaceous glands with what appeared to be a secondary follicular dystrophy. A second line, B6N(Cg)-Ppp1r9b^{tm1.1(KOMP)Vlcg}/J, had follicular dystrophy limited to many but not all mystacial vibrissae in heterozygous but not homozygous mutant mice, suggesting that this was a nonspecific background lesion. We discuss potential reasons for the low frequency of skin and adnexal phenotypes in mice from this project in comparison to those seen in human Mendelian diseases, and suggest alternative approaches to identification of human disease-relevant models.

Introduction

How many genes are involved in the normal ontogeny and physiology of the skin and its adnexal structures? How do they interact and how are they controlled? These questions, simple to articulate, have complex answers at best, but are predicated on our knowledge of gene function and more importantly the pleiotropic functions of genes, which can only be elucidated though experimentation and particularly through genetic manipulation. Faced with this challenge, we might initially search major databases such as Mouse Genome Informatics (MGI; http://www.informatics.jax.org/), Online Mendelian Inheritance in Man (https://www.omim.org/), or Orphanet (http://www.orpha.net/) and ask whether naturally-occurring or genetically engineered mutations show skin and skin-associated phenotypes; this can then be the beginning of a detailed investigation of gene expression, function and control. However, we only have experimentally-derived functional information for about half (11,973) of the protein-coding genes in the mouse as assessed by Gene Ontology annotation (MGI statistics; data accessed 19 Feb 2017) of the estimated 24,211 total, and while there are now many electronically inferred annotations for cellular location, process, and function for many more genes, these are not reliable predictors of the biological process in which genes may be involved. Critically, we have some (in many cases sparse) phenotype information from only 3,710 genes in Online Mendelian Inheritance in Man (accessed 19 Feb 2017). This is therefore greatly enhanced by gene/phenotype data from genetically variant mouse strains for around 9,493 mouse genes with human orthologs. The consequence of this is that although the mouse provides a great deal more functional information than the human, much of the genome remains biologically featureless, its “dark matter”, and it is only by mutating genes one by one that we can begin to build up a picture of their involvement in processes and structures, and their patterns of functional pleiotropy which are fundamentally unpredictable from sequence alone. An additional problem with mutant mice made in pursuit of hypothesis-driven science is that they are often made on highly variable or undefined genetic backgrounds, often for particular purposes, without systematic or standardized whole animal phenotyping, making it very difficult to compare phenotypes between mutant mice in any uniform way.

To address this problem on a large-scale basis, an international consortium was formed in 2007 to inactivate (“knockout”) all protein-coding genes in the mouse genome in embryonic stem cells using multiple gene targeting approaches on a uniform C57BL/6N genetic background [1, 2]. The second phase of this grand plan, the large-scale production and characterization of these mice, is now underway [3, 4]. The International Mouse Phenotyping Consortium was formed in 2011, composed of 17 Institutes across the world in 11 countries with 5 national funding agencies. This includes the NIH-supported contribution to these efforts, the Knockout Mouse Phenotyping Program, which consists of 3 groups; 1) The Jackson Laboratory, The 2) Baylor College of Medicine, The Wellcome Trust Sanger Institute and MRC Harwell Consortium, and 3) the University of California at Davis, The Toronto Center for Phenogenomics, Children’s Hospital Oakland Research Institute, and Charles River Laboratories Consortium). These consortia have generated over 5,000 strains of live mice using these embryonic stem cell lines and have recently transitioned to using CRISPR/Cas9-induced null alleles. Knockout lines are subjected to detailed, systematic evaluation (phenotyping). A majority of the phenotyping tests performed as part of these programs focus on physiological characterization as their primary goal [5]. While this approach generates an enormous amount of physiological data (clinical pathology, serology/hematology, behavioral data, etc.) [6, 7], histopathology, especially when done by highly trained experts focusing on specific organs, can often make the direct correlation quickly and accurately, as histologic evaluation by pathologists remains the definitive assay in human and veterinary clinical medicine for making most diagnoses [8, 9]. Furthermore, diseases of some organ systems are only truly defined at the gross clinical level followed by histopathology, special stains, and other tissue based tests, such as immunohistochemistry or electron microscopy. The skin, and its adnexa, is one such system.

While abnormalities of the skin and adnexa (eccrine glands, pilosebaceous unit, hair shaft, and nails) are easily seen by just looking at the mouse with the naked eye or with magnification (dissection or scanning electron microscope), detailed histopathology is still necessary to accurately define the abnormality in the skin and some lesions are only apparent microscopically. A primary high-throughput phenotyping strategy concentrates on wide coverage of a large number of parameters in a large number of mice and consequently is designed to pick up relatively clear-cut examples of phenodeviance.

Prior to the initiation of the current international efforts, both Lexicon and Deltagen undertook large-scale systematic mutagenesis and phenotyping projects, of which Lexicon’s used systematic histopathology as a routine phenotyping tool (see [10] for discussion) and generated histopathological analyses on nearly 5,000 mutant strains. Detailed descriptions of these strains are now in the process of being published in the literature [11, 12].

More recently, the Sanger Mouse Genetics Program reported a preliminary general histopathology workup of 50 randomly selected knockout lines yielding 2 with skin phenotypes, using single samples of dorsal skin from two males and two female mice [13]. This was complemented by a “special skin workup” as part of an extended primary phenotyping pipeline [14]. The histopathology protocol involved collecting one piece of dorsal skin, fixing, and processing it routinely to create hematoxylin and eosin stained slides. Nearly 800 mutant mouse skin samples were collected from 562 mutant lines in this high-throughput protocol. One and sometimes two slides with two or three serial sections of dorsal skin from one mouse in the study were provided. Mice were approximately 15 weeks of age. Only 23 lines (4.1%) had some sort of skin phenotype while all the others were normal, implying that many of the genes inactivated had no function in the skin; at least not evident from this screening protocol.

The International Mouse Phenotyping Consortium database currently (accessed 17 February 2017) shows that overall 2.22% of 3,199 lines show abnormalities of skin morphology and 5.35% of 2,971, abnormalities of the adnexa. This group reported no abnormalities of skin physiology in its database to date. A search of the Europhenome database, which overlaps with the International Mouse Phenotyping Consortium, shows 12 genes with annotations on skin morphology and physiology (excluding nociception; 27) of 563 genotypes derived from thesemutagenesis efforts (2.1%). The low number of skin phenotypes in these samples contrasts the frequent reporting of skin and adnexal phenotypes both in human Mendelian diseases and in the gathered data of MGI. Detailed examination of the MGI database shows that 1,069 of 9,045 targeted genes with phenotypes are annotated to either skin morphology or phenotypes; approximately 12%. In OMIM, 1,314/3,710 gene entries with phenotypic annotations are annotated to “abnormality of skin, HP:000095” or its children in the Human Phenotype Ontology database (35%) [15]. Assuming that we might expect skin and adnexal phenotypes in both humans and mice to result from loss of function of the same gene, the key question is where are the missing phenotypes? Even considering the exceptionally detailed phenotyping carried out in clinical practice, this is a large discrepancy. One possibility is that in the mouse, loss of function genes involved in the development or function of the skin and adnexa might be embryonic or neonatal lethal. However, this may also be an issue of detection; either through skewed phenotyping (i.e. not focused on the skin or not reported) or failure to detect phenotypes. There are projects associated with the US KnockOut Mouse Program that examine embryonic and perinatal lethality, yet none of them look beyond gross skin phenotype as reasons for early mortality [4]. From the reasoning above, we believe this problem can be addressed through careful targeted skin phenotyping.

The Jackson Laboratory is one of three sites in the United States generating knockout mutant mice. Histopathology is an optional parameter in the phenotyping pipeline, and thus is not performed and/or reported universally from individual centers. To better address finding skin lesions, representative retired breeder mice were received, histology was done on skin and nails from multiple sites, and slides were screened by a veterinary pathologist specializing in phenotyping skin mutants. Candidate models were subsequently reviewed by a physician pathologist specializing in dermatopathology. In so doing 169 novel mutant lines were reviewed and of those only one was found to have a relatively minor sebaceous gland abnormality.

Results

Skin, hair follicles, and nail units of both sexes were screened for lesions in 169 novel mutant lines of knockout mice generated at JAX (Table 1). Of these 116 (68.6%) were homozygous and 61 (36.1%) were heterozygous mutant mice. Four lines had both heterozygous and homozygous mice screened (2.4%). Of the heterozygous mice, 62.3% were pre-wean lethal and 11.5% were sub-viable, which is defined as yielding fewer than half of the expected number of homozygotes from an intercross (<12.5%). For these strains no homozygous mutant mice were evaluated. A total of 743 (2.2 average per line and sex) mice were necropsied and the skin and adnexa were evaluated histologically. Nine lines (two females and three males for heterozygotes only and three females and one male for homozygotes only) had only one sex evaluated.

Table 1. KOMP lines evaluated.

Clinical summary of 169 KOMP strains screened for skin, hair, and nail abnormalities.

No.	Strain	JR #	Mutant Allele	No. Mice	Age (days)	Genotype	Hair Follicle	Seba-ceous Gland	Prenatal Lethality	Sub-viable
1	B6N(Cg)	24041	4933427D14Rik^{tm1.1(KOMP)Vlcg}/J	2	138/161	HET
1	B6N(Cg)	24041	4933427D14Rik^{tm1.1(KOMP)Vlcg}/J	2	138/161	HET
2	B6N(Cg)	24902	Acsf2^{tm1.1(KOMP)Vlcg}/J	1	218	HOM
2	B6N(Cg)	24902	Acsf2^{tm1.1(KOMP)Vlcg}/J	3	218/222	HOM
3	C57BL/6NJ	25814	Adad1^em1J/J	2	272/325	HET				X
3	C57BL/6NJ	25814	Adad1^em1J/J	2	308/337	HET				X
4	C57BL/6NJ	25475	Adad2^em1J/J	2	193/199	HET
4	C57BL/6NJ	25475	Adad2^em1J/J	2	196/199	HET
5	B6N(Cg)	18592	Adora2b^{tm1.1(KOMP)Vlcg}/J	2	282	HOM
5	B6N(Cg)	18592	Adora2b^{tm1.1(KOMP)Vlcg}/J	2	165/223	HET
6	B6N(Cg)	18561	Ahrr^{tm1b(KOMP)Wtsi}/J	2	41/138	HOM
6	B6N(Cg)	18561	Ahrr^{tm1b(KOMP)Wtsi}/J	2	41/138	HOM
7	B6N(Cg)	25117	Akip1^{tm1.1(KOMP)Vlcg}/J	2	183 /189	HOM
7	B6N(Cg)	25117	Akip1^{tm1.1(KOMP)Vlcg}/J	2	183/189	HOM
8	B6N(Cg)	24334	Akr1b8^{tm1.1(KOMP)Vlcg}/J	2	125	HOM
8	B6N(Cg)	24334	Akr1b8^{tm1.1(KOMP)Vlcg}/J	2	125	HOM
9	B6N(Cg)	24656	Anapc15^{tm1.1(KOMP)Vlcg}/J	2	125/139	HET			X
9	B6N(Cg)	24656	Anapc15^{tm1.1(KOMP)Vlcg}/J	2	166	HET			X
10	B6N(Cg)	21510	Ap4e1^{tm1b(KOMP)Wtsi}/J	2	266	HOM
10	B6N(Cg)	21510	Ap4e1^{tm1b(KOMP)Wtsi}/J	2	198/359	HOM
11	B6N(Cg)	22304	Arhgef10^{tm1.1(KOMP)Vlcg}/J	1	95	HOM
11	B6N(Cg)	22304	Arhgef10^{tm1.1(KOMP)Vlcg}/J	3	95/99	HOM
12	B6N(Cg)	18607	Arrdc1^{tm1.1(KOMP)Vlcg}/J	3	180/213	HET
12	B6N(Cg)	18607	Arrdc1^{tm1.1(KOMP)Vlcg}/J	1	180	HET
13	B6N(Cg)	18657	Arsk^{tm1b(KOMP)Wtsi}/J	2	254	HOM
13	B6N(Cg)	18657	Arsk^{tm1b(KOMP)Wtsi}/J	1	254	HOM
14	B6N(Cg)	18660	Asb10^{tm2b(KOMP)Wtsi}/J	2	173	HOM
14	B6N(Cg)	18660	Asb10^{tm2b(KOMP)Wtsi}/J	2	120	HOM
15	B6N(Cg)	22101	Asf1a^{tm1b(KOMP)Wtsi}/2J	2	320/385	HET			X
15	B6N(Cg)	22101	Asf1a^{tm1b(KOMP)Wtsi}/2J	2	362/386	HET			X
16	B6N(Cg)	22305	Atrip^{tm1b(KOMP)Wtsi}/J	2	340	HET			X
16	B6N(Cg)	22305	Atrip^{tm1b(KOMP)Wtsi}/J	2	346	HET			X
17	B6N(Cg)	24979	Bbs7^{tm1b(EUCOMM)Wtsi}/J	2	191/192	HET				X
17	B6N(Cg)	24979	Bbs7^{tm1b(EUCOMM)Wtsi}/J	2	177/192	HET				X
18	B6N(Cg)	21751	Btg2^{tm1b(KOMP)Mbp}/2J	2	223	HOM
18	B6N(Cg)	21751	Btg2^{tm1b(KOMP)Mbp}/2J	2	204/352	HOM
19	B6N(Cg)	18653	Bzw2^{tm1b(KOMP)Mpb}/J	2	186	HOM
19	B6N(Cg)	18653	Bzw2^{tm1b(KOMP)Mpb}/J	2	226	HOM
20	B6N(Cg)	22307	C1qa^{tm1b(EUCOMM)Wtsi}/3J	2	308	HOM
20	B6N(Cg)	22307	C1qa^{tm1b(EUCOMM)Wtsi}/3J	2	308	HOM
21	B6N(Cg)	18562	C1qtnf5^{tm1.1(KOMP)Vlcg}/J	2	380/472	HOM
21	B6N(Cg)	18562	C1qtnf5^{tm1.1(KOMP)Vlcg}/J	2	443	HOM
22	B6N(Cg)	22098	C3^{tm1.1(KOMP)Vlcg}/J	2	128/219	HOM
22	B6N(Cg)	22098	C3^{tm1.1(KOMP)Vlcg}/J	2	128/225	HOM
23	B6N(Cg)	21282	C9^{tm1(KOMP)Vlcg}/J	1	198/206	HET
23	B6N(Cg)	21282	C9^{tm1(KOMP)Vlcg}/J	2	269	HET
24	B6N(Cg)	22418	Cacna2d3^{tm1b(KOMP)Wtsi}/J	2	239	HOM
24	B6N(Cg)	22418	Cacna2d3^{tm1b(KOMP)Wtsi}/J	2	239	HOM
25	B6N(Cg)	23393	Cast^{tm1.1(KOMP)Vlcg}/2J	2	111/171	HOM
25	B6N(Cg)	23393	Cast^{tm1.1(KOMP)Vlcg}/2J	2	111/171	HOM
26	B6N(Cg)	18111	Ccd20^{tm1(KOMP)Vlcg}/J	2	337/339	HET			X
27	B6N(Cg)	24349	Ccl26^{tm1.1(KOMP)Vlcg}/J	3	216/232/ 251	HOM
27	B6N(Cg)	24349	Ccl26^{tm1.1(KOMP)Vlcg}/J	1	157	HOM
28	B6N(Cg)	22016	Cd33^{tm1.1(KOMP)Vlcg}/J	4	147/321	HOM
28	B6N(Cg)	22016	Cd33^{tm1.1(KOMP)Vlcg}/J	4	176/321	HOM
29	B6N(Cg)	22335	Ceacam16^{tm1.1(KOMP)Wtsi}/J	4	100/207/ 227/285	HOM
29	B6N(Cg)	22335	Ceacam16^{tm1.1(KOMP)Wtsi}/J	4	100/207/ 273/285	HOM
30	B6N(Cg)	18836	Cenpo^{tm1b(KOMP)Mbp}/J	2	313/379	HET			X
30	B6N(Cg)	18836	Cenpo^{tm1b(KOMP)Mbp}/J	2	354/379	HET			X
31	B6N(Cg)	22819	Cers5^{tm2b(KOMP)Mbp}/J	2	206	HOM
31	B6N(Cg)	22819	Cers5^{tm2b(KOMP)Mbp}/J	2	206	HOM
32	B6N(Cg)	18603	Chn1^{tm1.1(KOMP)Vlcg}/J	2	244/247	HOM
32	B6N(Cg)	18603	Chn1^{tm1.1(KOMP)Vlcg}/J	2	244/246	HOM
33	B6N(Cg)	25118	Chp2^{tm1b(KOMP)Mbp}/J	3	149/210/ 211	HET
33	B6N(Cg)	25118	Chp2^{tm1b(KOMP)Mbp}/J	2	210/211	HET
34	B6N(Cg)	23317	Chtf18^{tm1b(KOMP)Wtsi}/J	2	300	HET				X
34	B6N(Cg)	23317	Chtf18^{tm1b(KOMP)Wtsi}/J	2	300	HET				X
35	B6N(Cg)	18631	Cinp^{tm1.1(KOMP)Vlcg}/J	2	126/136	HET			X
35	B6N(Cg)	18631	Cinp^{tm1.1(KOMP)Vlcg}/J	4	131/135/ 136	HET			X
36	B6N(Cg)	18587	Cited4^{tm1.1(KOMP)Vlcg}/J	2	140/183	HOM
36	B6N(Cg)	18587	Cited4^{tm1.1(KOMP)Vlcg}/J	2	140/183	HOM
37	B6N(Cg)	23666	Cldn19^{tm1.1(KOMP)Vlcg}/J	2	230/324	HOM
37	B6N(Cg)	23666	Cldn19^{tm1.1(KOMP)Vlcg}/J	2	230/255	HOM
38a	B6N(Cg)	18604	Col18a1^{tm1.1(KOMP)Vlcg}/J	2	193/208	HET
38a	B6N(Cg)	18604	Col18a1^{tm1.1(KOMP)Vlcg}/J	2	191/208	HET
38b	B6N(Cg)	18604	Col18a1^{tm1.1(KOMP)Vlcg}/J	2	322/350	HOM
38b	B6N(Cg)	18604	Col18a1^{tm1.1(KOMP)Vlcg}/J	2	322/350	HOM
39	B6N(Cg)	21485	Cp^{tm1b(KOMP)Wtsi}/J	2	184/326	HOM
39	B6N(Cg)	21485	Cp^{tm1b(KOMP)Wtsi}/J	2	271/326	HOM
40	B6N(Cg)	22015	Crym^{tm1b(KOMP)Wtsi}/J	2	161/198	HOM
40	B6N(Cg)	22015	Crym^{tm1b(KOMP)Wtsi}/J	2	161/198	HOM
41	B6N(Cg)	22096	Cyb5d1^{tm1.1(KOMP)Vlcg}/J	3	323/354	HOM
41	B6N(Cg)	22096	Cyb5d1^{tm1.1(KOMP)Vlcg}/J	1	355	HOM
42	B6N(Cg)	24035	Cyb5d2^{tm1.1(KOMP)Wtsi}/J	2	203	HOM
42	B6N(Cg)	24035	Cyb5d2^{tm1.1(KOMP)Wtsi}/J	2	203	HOM
43	B6N(Cg)	22777	Dbf4^{tm1b(KOMP)Wtsi}/J	2	137/155	HET			X
43	B6N(Cg)	22777	Dbf4^{tm1b(KOMP)Wtsi}/J	2	178	HET			X
44	B6N(Cg)	27202	Dbn1^{tm1a(KOMP)Wtsi}/J	2	274	HET				X
44	B6N(Cg)	27202	Dbn1^{tm1a(KOMP)Wtsi}/J	2	200/241	HET				X
45	B6N(Cg)	23604	Dcps^{tm1b(EUCOMM)Hmgu}/J	2	249	HET			X
45	B6N(Cg)	23604	Dcps^{tm1b(EUCOMM)Hmgu}/J	2	258	HET			X
46	B6N(Cg)	18632	Dnajb3^{tm1.1(KOMP)Vlcg}/J	2	209/240	HOM
46	B6N(Cg)	18632	Dnajb3^{tm1.1(KOMP)Vlcg}/J	2	202/240	HOM
47	B6N(Cg)	18580	Dnajb7^{tm1.1(KOMP)Vlcg}/J	1	250	HOM
47	B6N(Cg)	18580	Dnajb7^{tm1.1(KOMP)Vlcg}/J	2	268	HOM
48	B6N(Cg)	18597	Dnajb12^{tm1.1(KOMP)Vlcg}/J	2	156/243	HOM
49	B6N(Cg)	18616	Dnajc5g^{tm1.1(KOMP)Vlcg}/J	2	301	HOM
49	B6N(Cg)	18616	Dnajc5g^{tm1.1(KOMP)Vlcg}/J	2	164	HOM
50	B6N(Cg)	18637	Dnajc14^{tm1.1(KOMP)Vlcg}/J	4	136/177	HOM
50	B6N(Cg)	18637	Dnajc14^{tm1.1(KOMP)Vlcg}/J	4	117/177/ 352	HOM
51	B6N(Cg)	18633	Dnajc16^{tm1.1(KOMP)Vlcg}/J	1	122	HET				X
51	B6N(Cg)	18633	Dnajc16^{tm1.1(KOMP)Vlcg}/J	2	122	HET				X
52	B6N(Cg)	21508	Dnase1l2^{tm1.1(KOMP)Wtsi}/J	2	326	HOM
53	B6N(Cg)	26437	Enox1^{tm1.1(KOMP)Vlcg}/J	3	239	HET
53	B6N(Cg)	26437	Enox1^{tm1.1(KOMP)Vlcg}/J	1	266	HET
54	B6N(Cg)	23489	Ermp1^{tm1.1(KOMP)Vlcg}/J	4	167/201/ 220	HOM
54	B6N(Cg)	23489	Ermp1^{tm1.1(KOMP)Vlcg}/J	4	170/173/ 201	HOM
55	B6N(Cg)	24660	Espnl^{tm1b(KOMP)Wtsi}/2J	2	180	HOM
55	B6N(Cg)	24660	Espnl^{tm1b(KOMP)Wtsi}/2J	2	145	HOM
56	C57BL/6NJ	25733	Exoc4^em1J/J	4	121/123	HET			X
56	C57BL/6NJ	25733	Exoc4^em1J/J	2	121/123	HET			X
57	B6N(Cg)	18622	Exosc8^{tm1.1(KOMP)Vlcg}/J	2	379	HET			X
57	B6N(Cg)	18622	Exosc8^{tm1.1(KOMP)Vlcg}/J	2	429	HET			X
58	B6N(Cg)	18705	Fam161a^{tm1b(KOMP)Wtsi}/2J	2	209	HOM
58	B6N(Cg)	18705	Fam161a^{tm1b(KOMP)Wtsi}/2J	2	253/316	HOM
59	B6N(Cg)	22011	Far2^{tm2b(KOMP)Wtsi}/2J	2	184	HOM	dystrophy	hypo-plasia
59	B6N(Cg)	22011	Far2^{tm2b(KOMP)Wtsi}/2J	2	172/184	HOM	dystrophy	hypo-plasia
60	B6N(Cg)	22097	Foxo3^{tm1.1(KOMP)Vlcg}/J	2	148	HOM
60	B6N(Cg)	22097	Foxo3^{tm1.1(KOMP)Vlcg}/J	2	226	HOM
61	B6N(Cg)	21486	Ghr^{tm1b(KOMP)Wtsi}/3J	2	304/337	HET
61	B6N(Cg)	21486	Ghr^{tm1b(KOMP)Wtsi}/3J	2	304/337	HET
62	B6N(Cg)	19459	Ghrhr^{tm1.1(KOMP)Vlcg}/2J	1	292	HOM
62	B6N(Cg)	19459	Ghrhr^{tm1.1(KOMP)Vlcg}/2J	2	141	HOM
63	B6N(Cg)	18595	Ghsr^{tm1.1(KOMP)Vlcg}/J	4	143/153/ 370	HOM
63	B6N(Cg)	18595	Ghsr^{tm1.1(KOMP)Vlcg}/J	4	156/370	HOM
64	B6N(Cg)	18643	Gipc3^{tm1b(KOMP)Wtsi}/J	2	208	HOM
64	B6N(Cg)	18643	Gipc3^{tm1b(KOMP)Wtsi}/J	2	208	HOM
65	B6N(Cg)	18558	Glycam1^{tm1.1(KOMP)Vlcg}/J	1	180	HOM
65	B6N(Cg)	18558	Glycam1^{tm1.1(KOMP)Vlcg}/J	1	180	HOM
66	B6N(Cg)	24058	Gnb5^{tm1.1(KOMP)Vlcg}/J	2	174/185	HET				X
66	B6N(Cg)	24058	Gnb5^{tm1.1(KOMP)Vlcg}/J	2	186	HET				X
67	B6N(Cg)	18594	H1fx^{tm1.1(KOMP)Vlcg}/J	2	189	HOM
67	B6N(Cg)	18594	H1fx^{tm1.1(KOMP)Vlcg}/J	2	186/195	HOM
68	C57BL/6NJ	25319	Heyl^{tm1b(KOMP)Wtsi}/J	2	215	HOM
68	C57BL/6NJ	25319	Heyl^{tm1b(KOMP)Wtsi}/J	2	215	HOM
69	B6N(Cg)	24947	Hfe2^{tm1b(KOMP)Wtsi}/J	2	168/197	HOM
69	B6N(Cg)	24947	Hfe2^{tm1b(KOMP)Wtsi}/J	2	197/198	HOM
70	B6N(Cg)	24981	Hsf2^{tm1b(KOMP)Wtsi}/3J	2	172	HOM
70	B6N(Cg)	24981	Hsf2^{tm1b(KOMP)Wtsi}/3J	2	172	HOM
71	B6N(Cg)	22782	Hspb1^{tm1(KOMP)Vlcg}/2J	2	188	HET
72	B6N(Cg)	18619	Hspb2^{tm1.1(KOMP)Vlcg}/J	4	262/263/ 386	HOM
72	B6N(Cg)	18619	Hspb2^{tm1.1(KOMP)Vlcg}/J	4	262/263/ 386	HOM
73	B6N(Cg)	18618	Hspb3^{tm1.1(KOMP)Vlcg}/J	2	144/199	HOM
73	B6N(Cg)	18618	Hspb3^{tm1.1(KOMP)Vlcg}/J	2	144/199	HOM
74	B6N(Cg)	18617	Htr1a^{tm1.1(KOMP)Vlcg}/J	2	345	HOM
74	B6N(Cg)	18617	Htr1a^{tm1.1(KOMP)Vlcg}/J	2	137/308	HOM
75	B6N(Cg)	24347	Htr1d^{tm1.1(KOMP)Vlcg}/2J	2	165	HOM
75	B6N(Cg)	24347	Htr1d^{tm1.1(KOMP)Vlcg}/2J	2	165/226	HOM
76	B6N(Cg)	18615	Htr3b^{tm1.1(KOMP)Vlcg}/J	2	299	HOM
76	B6N(Cg)	18615	Htr3b^{tm1.1(KOMP)Vlcg}/J	2	299	HOM
77	B6N(Cg)	23606	Ift88^{tm1.1(KOMP)Vlcg}/J	2	350	HET			X
77	B6N(Cg)	23606	Ift88^{tm1.1(KOMP)Vlcg}/J	2	349/350	HET			X
78a	B6N(Cg)	21516	Igsf11^{tm1b(KOMP)Wtsi}/J	1	297	HET
78a	B6N(Cg)	21516	Igsf11^{tm1b(KOMP)Wtsi}/J	3	287/300	HET
78b	B6N(Cg)	21516	Igsf11^{tm1b(KOMP)Wtsi}/J	2	280/351	HOM
78b	B6N(Cg)	21516	Igsf11^{tm1b(KOMP)Wtsi}/J	2	280	HOM
79	B6N(Cg)	22303	Il12rb2^{tm1.1(KOMP)Vlcg}/J	4	168/206/ 264	HOM
79	B6N(Cg)	22303	Il12rb2^{tm1.1(KOMP)Vlcg}/J	4	168/206	HOM
80	B6N(Cg)	22005	Il24^{tm1.1(KOMP)Vlcg}/2J	4	64/165/194/214	HOM
80	B6N(Cg)	22005	Il24^{tm1.1(KOMP)Vlcg}/2J	4	64/165/194/214	HOM
81	B6N(Cg)	22388	Il6st^{tm1b(KOMP)Mbp}/J	2	180/210	HOM
81	B6N(Cg)	22388	Il6st^{tm1b(KOMP)Mbp}/J	2	210	HOM
82	B6N(Cg)	18655	Irf8^{tm1b(KOMP)Wtsi}/J	2	195	HOM
82	B6N(Cg)	18655	Irf8^{tm1b(KOMP)Wtsi}/J	2	171/195	HOM
83	B6N(Cg)	24055	Jam2^{tm1.1(KOMP)Mbp}/J	2	140	HOM
83	B6N(Cg)	24055	Jam2^{tm1.1(KOMP)Mbp}/J	2	140	HOM
84	B6N(Cg)	26066	Jmjd8^{tm1.1(KOMP)Vlcg}/J	3	269	HOM
84	B6N(Cg)	26066	Jmjd8^{tm1.1(KOMP)Vlcg}/J	1	269	HOM
85	B6N(Cg)	22821	Jup^{tm1.1(KOMP)Vlcg}/J	2	109/143	HET			X
85	B6N(Cg)	22821	Jup^{tm1.1(KOMP)Vlcg}/J	2	107/143	HET			X
86	B6N(Cg)	21140	Kcnh3^{tm1.1(KOMP)Vlcg}/2J	1	186	HOM
86	B6N(Cg)	21140	Kcnh3^{tm1.1(KOMP)Vlcg}/2J	2	186/288	HOM
87	B6N(Cg)	23603	Kif1b^{tm1b(KOMP)Wtsi}/2J	2	319	HET			X
87	B6N(Cg)	23603	Kif1b^{tm1b(KOMP)Wtsi}/2J	2	322	HET			X
88	B6N(Cg)	22085	Kif26b^{tm2b(KOMP)Wtsi}/J	2	323/446	HET			X
88	B6N(Cg)	22085	Kif26b^{tm2b(KOMP)Wtsi}/J	2	327/446	HET			X
89	B6N(Cg)	25381	Klk14^{tm1.1(KOMP)Vlcg}/J	2	115/161	HOM
89	B6N(Cg)	25381	Klk14^{tm1.1(KOMP)Vlcg}/J	2	115/152	HOM
90	B6N(Cg)	23318	Krt17^{tm1.1(KOMP)Vlcg}/2J	2	297/304	HET
90	B6N(Cg)	23318	Krt17^{tm1.1(KOMP)Vlcg}/2J	2	297/304	HET
91	B6N(Cg)	25383	Krt9^{tm1.1(KOMP)Vlcg}/J	2	210	HOM
91	B6N(Cg)	25383	Krt9^{tm1.1(KOMP)Vlcg}/J	2	210	HOM
92	B6N(Cg)	25518	Lipn^{tm1.1(KOMP)Vlcg}/J	4	188/189/ 195	HOM
93	B6N(Cg)	18563	Loxl1^{tm1.1(KOMP)Vlcg}/J	4	112/133/ 147	HET
93	B6N(Cg)	18563	Loxl1^{tm1.1(KOMP)Vlcg}/J	2	110	HET
94	B6N(Cg)	22094	Lpar6^{tm1.1(KOMP)Vlcg}/J	2	172/226	HOM
94	B6N(Cg)	22094	Lpar6^{tm1.1(KOMP)Vlcg}/J	2	172/213	HOM
95	B6N(Cg)	23021	Lrrc15^{tm1b(KOMP)Wtsi}/J	1	183	HOM
95	B6N(Cg)	23021	Lrrc15^{tm1b(KOMP)Wtsi}/J	1	48	HOM
96	B6N(Cg)	23024	Mdk^{tm1.1(KOMP)Mbp}/J	2	193	HOM
96	B6N(Cg)	23024	Mdk^{tm1.1(KOMP)Mbp}/J	2	193	HOM
97a	B6N(Cg)	22092	Mmp8^{tm1(KOMP)Vlcg}/J	2	213	HET
97b	B6N(Cg)	22092	Mmp8^{tm1.1(KOMP)Vlcg}/J	2	232/273	HOM
97b	B6N(Cg)	22092	Mmp8^{tm1.1(KOMP)Vlcg}/J	2	245/273	HOM
98	B6N(Cg)	18635	Mrps25^{tm1.1(KOMP)Vlcg}/J	2	303	HET			X
98	B6N(Cg)	18635	Mrps25^{tm1.1(KOMP)Vlcg}/J	2	303/364	HET			X
99	B6N(Cg)	24673	Myo7b^{tm1b(EUCOMM)Hmgu}/J	2	237/240	HOM
99	B6N(Cg)	24673	Myo7b^{tm1b(EUCOMM)Hmgu}/J	2	236/237	HOM
100	B6N(Cg)	23599	Nat1^{tm1.1(KOMP)Vlcg}/J	2	142/152	HOM
100	B6N(Cg)	23599	Nat1^{tm1.1(KOMP)Vlcg}/J	2	142/157	HOM
101	B6N(Cg)	18575	Ncald^{tm1.1(KOMP)Vlcg}/J	3	230/315	HOM
101	B6N(Cg)	18575	Ncald^{tm1.1(KOMP)Vlcg}/J	3	230/310/ 408	HOM
102	B6N(Cg)	18577	Nefh^{tm1.1(KOMP)Vlcg}/J	1	230	HOM
102	B6N(Cg)	18577	Nefh^{tm1.1(KOMP)Vlcg}/J	1	230	HOM
103	B6N(Cg)	18697	Nemf^{tm1b(EUCOMM)Hmgu}/J	2	78	HET			X
103	B6N(Cg)	18697	Nemf^{tm1b(EUCOMM)Hmgu}/J	2	78	HET			X
104	B6N(Cg)	24185	Nes^{tm1b(KOMP)Wtsi}/J	2	139/140	HOM
104	B6N(Cg)	24185	Nes^{tm1b(KOMP)Wtsi}/J	2	162/170	HOM
105	B6N(Cg)	18638	Nmrk2^{tm1.1(KOMP)Vlcg}/J	2	172/177	HOM
105	B6N(Cg)	18638	Nmrk2^{tm1.1(KOMP)Vlcg}/J	2	177/180	HOM
106	B6N(Cg)	18645	Nrcam^{tm2e.1(KOMP)Wtsi}/J	2	242/275	HOM
106	B6N(Cg)	18645	Nrcam^{tm2e.1(KOMP)Wtsi}/J	2	242/275	HOM
107	B6N(Cg)	24899	Nsun7^{tm1.1(KOMP)Vlcg}/J	3	183/190	HET
107	B6N(Cg)	24899	Nsun7^{tm1.1(KOMP)Vlcg}/J	1	183	HET
108	B6N(Cg)	24233	Nxn^{tm1b(EUCOMM)Wtsi}/J	2	232/236	HET			X
108	B6N(Cg)	24233	Nxn^{tm1b(EUCOMM)Wtsi}/J	2	236	HET			X
109	B6N(Cg)	24036	Orc1^{tm1b(KOMP)Wtsi}/J	2	185	HET			X
109	B6N(Cg)	24036	Orc1^{tm1b(KOMP)Wtsi}/J	2	185/206	HET			X
110	B6N(Cg)	22338	Osm^{tm1b(KOMP)Wtsi}/J	4	198/270/ 358/376	HOM
110	B6N(Cg)	22338	Osm^{tm1b(KOMP)Wtsi}/J	4	198/270/ 358	HOM
111	B6N(Cg)	23058	Parp8^{tm1.1(KOMP)Wtsi}/J	2	202	HOM
111	B6N(Cg)	23058	Parp8^{tm1.1(KOMP)Wtsi}/J	2	202	HOM
112	B6N(Cg)	21613	Parp16^{tm1.1(KOMP)Mbp}/2J	2	288	HOM
112	B6N(Cg)	21613	Parp16^{tm1.1(KOMP)Mbp}/2J	2	265	HOM
113	B6N(Cg)	22341	Pcsk5^{tm1b(KOMP)Mbp}/J	2	306/321	HET			X
113	B6N(Cg)	22341	Pcsk5^{tm1b(KOMP)Mbp}/J	2	346	HET			X
114	B6N(Cg)	24330	Pnmt^{tm1.1(KOMP)Vlcg}/J	4	120/140/ 152/182	HOM
114	B6N(Cg)	24330	Pnmt^{tm1.1(KOMP)Vlcg}/J	3	140/182	HOM
115a	B6N(Cg)	18609	Ppp1r9b^{tm1.1(KOMP)Vlcg}/J	4	139/282	HET	vibrissa
115a	B6N(Cg)	18609	Ppp1r9b^{tm1.1(KOMP)Vlcg}/J	2	139/330	HET	vibrissa
115b	B6N(Cg)	18609	Ppp1r9b^{tm1.1(KOMP)Vlcg}/J	2	321	HOM
115b	B6N(Cg)	18609	Ppp1r9b^{tm1.1(KOMP)Vlcg}/J	2	114/137	HOM
116	B6N(Cg)	22099	Prep^{tm1b(KOMP)Wtsi}/J	2	328/350	HET			X
116	B6N(Cg)	22099	Prep^{tm1b(KOMP)Wtsi}/J	2	350	HET			X
117a	B6N(Cg)	18576	Prom2^{tm1.1(KOMP)Vlcg}/J	3	89	HET
117a	B6N(Cg)	18576	Prom2^{tm1.1(KOMP)Vlcg}/J	1	89	HET
117b	B6N(Cg)	18576	Prom2^{tm1.1(KOMP)Vlcg}/J	2	403	HOM
117b	B6N(Cg)	18576	Prom2^{tm1.1(KOMP)Vlcg}/J	2	437	HOM
118	B6N(Cg)	24025	Psen1^{tm1.1(KOMP)Vlcg}/J	2	225/240	HET			X
118	B6N(Cg)	24025	Psen1^{tm1.1(KOMP)Vlcg}/J	2	240	HET			X
119	B6N(Cg)	23667	Pycr1^{tm1.1(KOMP)Vlcg}/J	2	165/251	HOM
119	B6N(Cg)	23667	Pycr1^{tm1.1(KOMP)Vlcg}/J	2	165/251	HOM
120	B6N(Cg)	22387	Rab5a^{tm1b(KOMP)Wtsi}/J	4	222/230	HOM
120	B6N(Cg)	22387	Rab5a^{tm1b(KOMP)Wtsi}/J	4	222/227/ 230	HOM
121	B6N(Cg)	22389	Rab24^{tm1(KOMP)Wtsi}/J	2	179	HET
121	B6N(Cg)	22389	Rab24^{tm1(KOMP)Wtsi}/J	2	243	HET
122	B6N(Cg)	23422	Rab27b^{tm1b(KOMP)Wtsi}/J	2	127/129	HOM
122	B6N(Cg)	23422	Rab27b^{tm1b(KOMP)Wtsi}/J	2	133/159	HOM
123	B6N(Cg)	24188	Rab34^{tm1b(EUCOMM)Hmgu}/J	2	328	HET			X
123	B6N(Cg)	24188	Rab34^{tm1b(EUCOMM)Hmgu}/J	2	328	HET			X
124	B6N(Cg)	23664	Rab36^{tm1b(KOMP)Wtsi}/J	2	96/255	HOM
124	B6N(Cg)	23664	Rab36^{tm1b(KOMP)Wtsi}/J	2	96/255	HOM
125	B6N(Cg)	23059	Rab43^{tm1.1(KOMP)Vlcg}/J	2	130	HOM
125	B6N(Cg)	23059	Rab43^{tm1.1(KOMP)Vlcg}/J	2	130	HOM
126	B6N(Cg)	26882	Rabac1^{tm1.1(KOMP)Vlcg}/J	2	118	HOM
126	B6N(Cg)	26882	Rabac1^{tm1.1(KOMP)Vlcg}/J	2	118	HOM
127	B6N(Cg)	25849	Rbpj^{tm1b(EUCOMM)Hmgu}/J	2	298	HET			X
127	B6N(Cg)	25849	Rbpj^{tm1b(EUCOMM)Hmgu}/J	2	255	HET			X
128	B6N(Cg)	18572	Resp18^{tm1.1(KOMP)Vlcg}/J	1	231	HOM
128	B6N(Cg)	18572	Resp18^{tm1.1(KOMP)Vlcg}/J	2	231	HOM
129	B6N(Cg)	24674	Rhbdl2^{tm1b(KOMP)Wtsi}/J	2	138	HOM
129	B6N(Cg)	24674	Rhbdl2^{tm1b(KOMP)Wtsi}/J	2	138	HOM
130	B6N(Cg)	21750	Rilpl2^{tm1b(KOMP)Wtsi}/J	4	150/198/ 287	HOM
130	B6N(Cg)	21750	Rilpl2^{tm1b(KOMP)Wtsi}/J	4	242/287/ 295	HOM
131	B6N(Cg)	21511	Rnf10^{tm1b(KOMP)Wtsi}/J	2	222	HOM
131	B6N(Cg)	21511	Rnf10^{tm1b(KOMP)Wtsi}/J	2	228	HOM
132	B6N(Cg)	26493	Rps6kl1^{tm1.1(KOMP)Vlcg}/J	2	242	HOM
132	B6N(Cg)	26493	Rsp6kl1^{tm1.1(KOMP)Vlcg}/J	2	242	HOM
133	B6N(Cg)	23136	Sdha^{tm2b(KOMP)Wtsi}/2J	2	343/393	HET			X
133	B6N(Cg)	23136	Sdha^{tm2b(KOMP)Wtsi}/2J	2	393/396	HET			X
134	B6N(Cg)	24038	Sgta^{tm1b(KOMP)Mbp}/J	2	250/254	HET
134	B6N(Cg)	24038	Sgta^{tm1b(KOMP)Mbp}/J	2	250	HET
135	B6N(Cg)	21142	Sh3tc2^{tm1b(KOMP)Wtsi}/J	2	221	HOM
135	B6N(Cg)	21142	Sh3tc2^{tm1b(KOMP)Wtsi}/J	1	227	HOM
136	B6N(Cg)	23511	Ska2^{tm1b(KOMP)Wtsi}/J	2	228	HET			X
136	B6N(Cg)	23511	Ska2^{tm1b(KOMP)Wtsi}/J	2	231	HET			X
137	B6N(Cg)	23414	Slc25a35^{tm1.1(KOMP)Vlcg}/J	2	179	HOM
137	B6N(Cg)	23414	Slc25a35^{tm1.1(KOMP)Vlcg}/J	2	179	HOM
138a	B6N(Cg)	22780	Sorbs2^tm1(KOMP)Mbp/J	1	86	HET
138a	B6N(Cg)	22780	Sorbs2^tm1(KOMP)Mbp/J	3	86/199	HET
138b	B6N(Cg)	22780	Sorbs2^{tm1.1(KOMP)Mbp}/J	1	159	HOM
138b	B6N(Cg)	22780	Sorbs2^{tm1.1(KOMP)Mbp}/J	2	159/160	HOM
139	B6N(Cg)	23020	Sox18^{tm1.1(KOMP)Vlcg}/J	2	178/226	HOM
139	B6N(Cg)	23020	Sox18^{tm1.1(KOMP)Vlcg}/J	2	161/165	HOM
140	B6N(Cg)	26095	Spag4^{tm1.1(KOMP)Mbp}/J	2	161	HOM
140	B6N(Cg)	26095	Spag4^{tm1.1(KOMP)Mbp}/J	1	59	HOM
141	B6N(Cg)	18820	Sptssa^{tm1b(KOMP)Wtsi}/J	2	287/292	HET			X
141	B6N(Cg)	18820	Sptssa^{tm1b(KOMP)Wtsi}/J	2	327	HET			X
142	B6N(Cg)	24184	Srd5a3^{tm1b(EUCOMM)Wtsi}/J	2	258/265	HET			X
142	B6N(Cg)	24184	Srd5a3^{tm1b(EUCOMM)Wtsi}/J	2	265	HET			X
143	B6N(Cg)	21115	Stag3^{tm1e.1(KOMP)Wtsi}/2J	1	234	HOM
143	B6N(Cg)	21115	Stag3^{tm1e.1(KOMP)Wtsi}/2J	2	104/275	HOM
144	B6N(Cg)	18611	Stk16^{tm1.1(KOMP)Vlcg}/J	2	105	HET			X
144	B6N(Cg)	18611	Stk16^{tm1.1(KOMP)Vlcg}/J	2	66/105	HET			X
145	B6N(Cg)	18611	Stk16^{tm1.1(KOMP)Vlcg}/J	2	39	HOM
145	B6N(Cg)	18611	Stk16^{tm1.1(KOMP)Vlcg}/J	2	39	HOM
146	B6N(Cg)	22781	Stx16^{tm1b(KOMP)Wtsi}/J	1	301	HOM
146	B6N(Cg)	22781	Stx16^{tm1b(KOMP)Wtsi}/J	2	301/320	HOM
147	B6N(Cg)	22784	Sycp3^{tm1(KOMP)Vlcg}/J	2	110	HET
147	B6N(Cg)	22784	Sycp3^{tm1(KOMP)Vlcg}/J	2	183	HET
148	B6N(Cg)	22784	Sycp3^{tm1.1(KOMP)Vlcg}/J	4	197/214/ 274/279	HOM
148	B6N(Cg)	22784	Sycp3^{tm1.1(KOMP)Vlcg}/J	4	165/202/ 274	HOM
149	B6N(Cg)	23665	Syn3^{tm1.1(KOMP)Vlcg}/J	2	267/270	HOM
149	B6N(Cg)	23665	Syn3^{tm1.1(KOMP)Vlcg}/J	2	267/270	HOM
150	B6.129S1	24694	Tgm2^tm1Rmgr/J	2	230/234	HOM
150	B6.129S1	24694	Tgm2^tm1Rmgr/J	2	203/234	HOM
151	B6N(Cg)	24406	Timm22^{tm1b(KOMP)Wtsi}/J	2	181/192	HET			X
151	B6N(Cg)	24406	Timm22^{tm1b(KOMP)Wtsi}/J	2	193	HET			X
152	B6N(Cg)	18639	Timp3^{tm1.1(KOMP)Vlcg}/J	3	210/278/ 292	HOM
152	B6N(Cg)	18639	Timp3^{tm1.1(KOMP)Vlcg}/J	1	210	HOM
153	B6N(Cg)	18629	Tmem136^{tm1.1(KOMP)Vlcg}/J	2	254/274	HOM
153	B6N(Cg)	18629	Tmem136^{tm1.1(KOMP)Vlcg}/J	2	187/274	HOM
154	B6N(Cg)	23060	Tmem151b^{tm1b(KOMP)Wtsi}/J	4	219/221/ 288	HOM
154	B6N(Cg)	23060	Tmem151b^{tm1b(KOMP)Wtsi}/J	4	202/288	HOM
155	B6N(Cg)	26542	Tpcn1^{tm1.1(KOMP)Vlcg}/J	3	174	HOM
155	B6N(Cg)	26542	Tpcn1^{tm1.1(KOMP)Vlcg}/J	1	174	HOM
156	B6N(Cg)	21752	Tpgs2^{tm1.1(KOMP)Vlcg}/J	2	156	HOM
156	B6N(Cg)	21752	Tpgs2^{tm1.1(KOMP)Vlcg}/J	2	156	HOM
157	B6N(Cg)	18606	Tprn^{tm1.1(KOMP)Vlcg}/J	2	270	HOM
157	B6N(Cg)	18606	Tprn^{tm1.1(KOMP)Vlcg}/J	2	141/293	HOM
158	B6N(Cg)	18553	Trip13^{tm1.1(KOMP)Vlcg}/J	4	181/196/ 239	HOM
158	B6N(Cg)	18533	Trip13^{tm1.1(KOMP)Vlcg}/J	4	41/181/321	HOM
159	B6N(Cg)	21483	Tspan18^{tm1.1(KOMP)Vlcg}/J	2	263	HOM
159	B6N(Cg)	21483	Tspan18^{tm1.1(KOMP)Vlcg}/J	2	263	HOM
160	C57BL/6NJ	26909	Ttll10^em1J/J	2	200/229	HOM
160	C57BL/6NJ	26909	Ttll10^em1J/J	2	196/229	HOM
161	B6N(Cg)	22817	Ube2c^{tm1.1(KOMP)Wtsi}/J	2	249/270	HET			X
161	B6N(Cg)	22817	Ube2c^{tm1.1(KOMP)Wtsi}/J	2	287	HET			X
162	B6N(Cg)	24027	Ube2m^{tm1.1(KOMP)Wtsi}/2J	2	484	HET			X
162	B6N(Cg)	24027	Ube2m^{tm1.1(KOMP)Wtsi}/2J	2	484	HET			X
163	B6N(Cg)	23019	Vsig8^{tm1.1(KOMP)Mbp}/J	1	116	HOM
163	B6N(Cg)	23019	Vsig8^{tm1.1(KOMP)Mbp}/J	1	44	HOM
164	B6N(Cg)	23660	Zbtb4^{tm1.1(KOMP)Vlcg}/J	2	267	HOM
164	B6N(Cg)	23660	Zbtb4^{tm1.1(KOMP)Vlcg}/J	2	144/267	HOM
165	B6N(Cg)	18620	Zdhhc11^{tm1.1(KOMP)Vlcg}/J	4	127	HET
166	B6N(Cg)	24091	Zfp346^{tm1.1(KOMP)Vlcg}/J	2	194/202	HET				X
166	B6N(Cg)	24091	Zfp346^{tm1.1(KOMP)Vlcg}/J	2	194/219	HET				X
167	B6N(Cg)	23023	Zfp42^{tm1b(KOMP)Mbp}/J	2	151	HOM
167	B6N(Cg)	23023	Zfp42^{tm1b(KOMP)Mbp}/J	2	145	HOM
168	B6N(Cg)	24057	Zfp961^{tm1.1(KOMP)Mbp}/J	2	179/226	HOM
168	B6N(Cg)	24057	Zfp961^{tm1.1(KOMP)Mbp}/J	2	179/226	HOM
169a	C57BL/6NJ	22366	Zzef1^{tm2(KOMP)Vlcg}/J	2	198/206	HET
169a	C57BL/6NJ	22366	Zzef1^{tm2(KOMP)Vlcg}/J	2	269	HET
169b	B6N(Cg)	22366	Zzef1^{tm2.1(KOMP)Vlcg}/J	1	103	HOM
169b	B6N(Cg)	22366	Zzef1^{tm2.1(KOMP)Vlcg}/J	2	95/352	HOM
	Females
	Males
	Strain not listed as B6N(Cg)
HET	Heterozygous mutant mice
HOM	Homozygous mutant mice
	Normal skin and adnexa
	Lesion in skin or adnexa
X	Prenatal lethal or subviable phenotype
	Clinically normal phenotype

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Nonspecific, sporadic lesions were found in many of the mice in this study. Most commonly these were ruptured hair follicles with hair shaft fragments free in the dermis or hypodermal fat layer surrounded by a variety of inflammatory cell types, usually neutrophils around the hair shaft and a layer of macrophages (trichogranuloma, Fig 1). These were often solitary and not consistently found in all the mice within a mutant line, which was a primary criterion to differentiate these lesions from gene mutation associated phenotypes. Other mice had follicular dystrophy consistent with C57BL/6 alopecia and dermatitis, a low frequency, strain-specific skin disease resembling human central centrifugal cicatricial alopecia [16].

Fig 1 — Incidental lesions were not consistently found in all mice of a line. Lesions included solitary tail skin hair follicle with infundibular plugging (A-C). Truncal hair follicle infundibular plugging, rupture below the level of the sebaceous gland, follicle filled with neutrophils and surrounded by neutrophils and macrophages (D-F). Sporadic skin lesions found in many mice examined included isolated, one or two, ruptured hair follicles in the dermis where the free hair shaft fragments were engulfed by granulomas and surrounded by neutrophils (G-I). Hematoxylin and eosin stain, bar size indicated in each figure.

Mice with null mutations in their fatty acyl CoA reductase 2 (Far2^{tm2b(KOMP)Wtsi}/2J) gene were found to consistently develop focal alopecia as adults on the dorsal skin extending distally from the base of the ears (Fig 2). Histologically, in areas where the skin appeared to be relatively normal there were minor lesions affecting the pilosebaceous and Meibomian (eyelids) glands that could be easily over-looked. Other modified sebaceous glands (Zymbal, clitoral, or preputial glands) were not examined in this study. The sebaceous glands superficially appeared to be normal but within the sebaceous duct the sebocytes became brightly eosinophilic and, when these holocrine cells ruptured, their secretion remained present and clumped. By contrast, normal sebocytes were slightly basophilic with fine, uniformly sized vacuoles in their cytoplasm. When the normal cells ruptured there was no evidence of their cytoplasmic content within the follicular infundibulum or on the skin surface (Fig 3). In areas of alopecia, the epidermis was mildly acanthotic with orthokeratotic hyperkeratosis associated with areas of follicular dystrophy and rupture and an associated foreign body inflammatory response that would lead to follicular scarring, a form of cicatricial alopecia (Fig 4). Skin surface lipidomic and time course studies are in progress to better define these changes. Further, non-integument phenotypes affecting lipid metabolism, behavior, immunity, and the cardiovascular system have been reported for this strain, and can be found on the IMPC database website (http://www.mousephenotype.org/data/genes/MGI:2687035#section-associations).

Fig 2 — Both are 15 week old females. Note the alopecia extending distally from the base of the ears.

Fig 3 — Most late stage anagen hair follicles in B6N(Cg)-*Far2*^{tm2b(KOMP)Wtsi}/2J mutant mice, as in this 184 day old male, were relatively normal and produced a normal hair shaft (A). However, the sebaceous glands were mildly hypoplastic with brightly eosinophilic mature sebocytes that did not immediately rupture as they entered the infundibulum (C). As in other mouse mutations that cause hypoplasia of sebaceous glands, the hair shafts in these mice did not exit the follicular ostium resulting in perforation of the root sheaths, release of the hair shaft into the dermis, and an inflammatory reaction (B). This healed by follicular scarring, or cicatricial alopecia. By contrast, aged and sex matched B6N(Cg) controls rarely exhibited ruptured follicles (D). Sebaceous glands varied in size with the hair cycle, which was normal, and sebocytes had light pink cytoplasm that becomes very pale as the cells matured and abruptly ruptured into the duct. (E). Hematoxylin and eosin stain, bar size indicated in each figure.

Fig 4 — In areas of alopecia, the B6N(Cg)- *Far2*^{tm2b(KOMP)Wtsi}/2J mutant skin was mildly acanthotic and orthokeratotic, most likely secondary to the abnormal secretions of the sebaceous glands. The infundibulum was mildly dilated (A, B). Follicles with follicular dystrophy (C, D) and with pigment cast (E) were identified occasionally to frequently. Such ruptured follicles with granulomatous inflammation eventually lead to follicular scarring, a form of cicatricial alopecia. Hematoxylin and eosin stain, bar size indicated in each figure.

Heterozygous mutant mice for the protein phosphatase 1, regulatory subunit 9B (Ppp1r9b^{tm1.1(KOMP)Vlcg}/J) gene had between one and five vibrissae hair shafts undergoing degenerative changes (follicular dystrophy) in each of three females and two males examined. Each section contained 10–15 vibrissae follicles (Fig 5). Only mystacial vibrissae were affected, not the periorbital or lower leg vibrissae. No other hair types were affected. This change was not found in two female and two male homozygous mutants suggesting that this was a nonspecific background lesion, as vibrissae follicular dystrophy was also seen occasionally in many mouse lines but only in one or two follicles and not consistently in all vibrissae in any section.

Fig 5 — B6N(Cg)-*Ppp1r9b*^{tm1.1(KOMP)Vlcg}/J, had follicular dystrophy limited to many but not all mystacial vibrissae in heterozygous but not homozygous mutant mice suggesting this is an incidental, albeit relatively frequent lesion. Boxed areas are enlarged as indicated by the arrows. Pigment casts are identified in all dystrophic hair follicles (B, F, and H). Hematoxylin and eosin stain, bar size indicated in each figure.

The skin and nail sections from almost all of the mice in these studies were clinically and histologically normal.

Discussion

We have investigated the apparent deficiency of skin and adnexal phenotypes in a random set of strains from a systematic, genome-wide, knockout study using histopathological phenotyping. We report here that numbers detected confirm an earlier study [14] which also showed low numbers of detectable skin phenotypes, suggesting that ascertainment is not the cause of the “missing phenotypes”.

Of over 100 homozygous mutants screened, only one line, Far2^{tm2b(KOMP)Wtsi}/2J, had lesions affecting sebaceous glands with what appeared to be a secondary follicular dystrophy, which probably progresses to a form of cicatricial alopecia as seen with a number of other mouse mutations with abnormalities of the sebaceous glands [17]. A rare autosomal recessive neurological disease without hair abnormalities was reported for humans with mutations in FAR1 [18]. While the biochemistry of these two proteins, FAR1 and FAR2, is known no disease is currently associated with mutations in FAR2 in humans (http://omim.org/entry/616156?search=Far2&highlight=far2). However, Far2 transcripts were reported in the eyelid and skin, presumably localized in the Meibomian and sebaceous glands, respectively [19]. Information obtained from phenotyping these mice will be useful to help assess sebaceous gland abnormalities in humans as not all genes that are expressed in sebaceous glands result in sebaceous gland abnormalities or clinically obvious hair problems, such as mouse null mutations in Soat1 [20, 21]. Detailed studies are in progress to define that pathogenesis of the follicular dystrophy and to match it with a specific human disease in these Far2 mutant mice.

A second line, Ppp1r9b^{tm1.1(KOMP)Vlcg}/J, had follicular dystrophy limited to many but not all vibrissae in heterozygous but not homozygous mutant mice suggesting this was a nonspecific background problem. A targeted mutant mouse line made for this gene (Ppp1r9b^tm1Jfe) was reported to only have neurological abnormalities [22, 23]. As the vibrissae are an important somatosensory organ in the mouse [24] this structure should be carefully evaluated in such phenotyping studies.

The International Mouse Phenotyping Consortium has provided a unique, internally consistent dataset with genome-wide coverage and has yielded a great deal of valuable information on gene function. At the outset anticipation was high that systematic creation of null mice for all known protein coding genes would yield large amounts of information on the pathobiology of human disease. The high frequency of embryonic and neonatal lethal lines [4], consistent with previous estimates [6], was not a surprise, but potentially limits the value of these mice for detailed investigation of adult phenotypes and diseases which will require carefully designed conditional strategies.

The relatively low frequency of skin phenotypes in the International Mouse Phenotyping Consortium mice found in this and an earlier study [11] was surprising given the frequency expected from global surveys of MGI and OMIM discussed above. The histopathological survey presented here was very detailed focusing on the skin, and we believe this supports the assertion that the “missing phenotypes” are not due to lack of ascertainment, at least in the random sample selected. The remaining possibilities are that genes involved in skin morphogenesis and physiology are overrepresented in those essential for embryogenesis, the C57BL6N background is failing to show phenotypes, or that the complete removal of gene function in the mouse knockouts have more severe functional consequences than the point mutations, regulatory lesions, and insertions/deletions (indels) that constitute most disease-causing mutations in humans. Protein truncating variants which provide the greatest likelihood of complete functional loss (“knockouts”) are very rare in the human population [25].

In the current study, 74% of the heterozygous lines were pre-wean lethal or sub-viable as homozygotes (that were not examined) presenting the possibility that mutants having skin phenotypes might also be embryonic essential genes. While it is possible to argue that essential genes should never show up as disease genes in a population due to negative selection, there is increasing evidence that essential genes are often ‘disease genes”–i.e. carry mutations that are associated with abnormal or disease phenotypes. This is by no means a settled conclusion however. The relationship between gene essentiality and disease association is discussed in detail in Cooper et al. [25].

Studies conducted around a decade ago, based on a limited gene set, suggested that mouse genes that are essential—i.e., genes related to more severe phenotypes in mouse, are more likely to be human disease genes, but that embryonic lethals were less likely [26]. However, a more recent study strongly suggested that about 30% of human disease genes (from OMIM) have a mouse ortholog with a lethal knockout phenotype (673/1965) [27]. The discrepancies here reflect to an extent the methodology used to calculate the association and the inclusion or exclusion of genes with no phenotypic data in the study by Park et al. [26]. This association will certainly stand further scrutiny now that more data are available, but does not explain why the IMPC/KOMP strains show fewer skin phenotypes than the bulk of the loss-of-function allele carrying strains in MGI. Core analysis carried out on Ingenuity^™ using the lists of lethal essential genes from supplementary table 3 of Dickinson et al. [4] shows no significant enrichment of annotations to dermatological disease or skin development, mitigating against the possibility that genes associated with skin disease are overrepresented in the essential segment of the genome. A working conclusion, therefore, is that either there is a problem with the strain background in this resource showing full expressivity of mutations affecting the skin, or that we have so far insufficient coverage of the genome using high-resolution phenotyping to account for the “missing phenotypes” from the International Mouse Phenotyping Consortium program. Of concern is the observation that most human disease associated mutations are hypomorphic alleles which, in the event of lethal homozygosity of complete KOs, are much more likely to present a phenotype compatible with survival. The potential for missing these with a complete loss of function strategy is significant, especially when examining completely novel genes where there are no existing experimental or inferred annotations. The power of looking for skin phenotypes using a strategy such as that developed by Beutler et al. [28] using ENU mutagenesis is very persuasive. Currently Mutagenetix (https://mutagenetix.utsouthwestern.edu/home.cfm), accessed 1 Mar 2017) shows 49 genes with skin associated phenotypes recovered as phenotypic mutations from a total of 262 phenotyped genes (119 of 498 mapped mutations) and should be considered in the future along with full characterization of spontaneous and ENU generated mutations in large collections.

Materials and methods

Generation of mutant mice

Embryonic stem cell lines

Initially mice included in the program were produced from embryonic stem cell lines acquired from repositories (https://www.komp.org/ or http://www.eummcr.org/). These resources comprised two major allele types, the Velocigene “definitive null” allele produced by the Regeneron Corporation (Vlcg) [1] and the “knockout first, conditional ready” design produced by the Wellcome Trust Sanger Centre and partners (Sanger) [2]. The Vlcg alleles delete the entire coding sequence of the target gene, including all introns, while inserting a LacZ reporter cassette. The neo resistance cassette is flanked by loxP sites for removal by cre-recombinase. The Sanger allele is a multifunctional design. In its native form, the strong splice acceptor typically generates a null allele. When bred to Flp-expressing mice, the entire 5’ LacZ and neo cassette are removed leaving a loxP-flanked critical exon for conditional mutagenesis. When bred to cre-recombinase transgenic mice, this allele is converted into a “definitive null” which retains the 5’ LacZ cassette and removes the critical exon and neo marker.

All animal work was approved by The Jackson Laboratory Animal Care and Use Committee (approval number 07005 "Mouse Models for Dermatology Research”).

Generating live mice

Following importation at The Jackson Laboratory, clones are injected into blastocyst hosts and resulting chimeras are bred to C57BL/6NJ (STOCK#5304) or C57BL/6NJ-Tyr^em3J/GrsrJ (STOCK#21999; to select for coat color transmission) inbred mice (see Table 1) to assure isogenicity of the resource. Germline transmitting (GLT) clones are quality confirmed (both Vlcg and Sanger) for targeting. Following GLT, mice are bred to congenic cre-recombinase transgenic mice (B6N.Cg-Tg(Sox2-cre)1Amc/J; STOCK#14094) to remove the neo cassette and to generate a definitive null allele, generating the tm1.1 allele (Vlcg and non-conditional Sanger clones) and tm1b allele (from conditional-ready Sanger clones). Four lines were generated by CRISPR/Cas9 mutagenesis, using standard approaches [29] to generate small deletions in the early part of the transcript. These mice are expanded, quality control-confirmed, and intercrossed to generate knockout mice. Approximately 30% of lines display lethality or sub-lethality (defined as yielding less than 12.5% homozygous mice) [4]. For these strains, heterozygous mice were phenotyped. For other strains, homozygous breeder pairs established for colony maintenance used in this study when they became retired breeders to maximize chances of finding adult onset diseases.

Systematic analysis of mutant mice

Animal logistics

Two female and two male retired breeder mice were provided from The Jackson Laboratory knockout mouse production program (Table 1). Homozygous lethal embryos and perinatal lethal mouse lines were specifically excluded from these studies. In these cases, heterozygous animals were aged and evaluated. All lines, whether Vlcg definitive null or Sanger conditional-ready mice were evaluated post-cre excision, which removed the neo cassette and heterologous promoter and in the case of Sanger alleles also removing the critical exon to create a definitive null allele (Vlcg tm1.1 and Sanger tm1b). All mice had skin, hair, and nails collected (see below).

Necropsy procedures, slide generation, and histopathologic analyses

As the skin is anatomically and molecularly distinct throughout the body in both mice and humans [30, 31] and diseases can be restricted to specific anatomic sites, skin was collected from several areas. As dorsal and ventral trunk skin are histologically similar in the mouse, they were collected with other anatomically identifiable tissues and put into respective cassettes: 1) dorsal skin and ear skin; 2) ventral skin, eyelid skin, muzzle skin; 3) digits to include the foot pads and nail unit; and 4) cross and longitudinal sections of the tail. This allows for evaluation of all major hair follicle types, mucocutaneous junctions, the nail unit, as well as the footpad (including eccrine glands) [32]. Detailed protocols on skin removal, orientation, and processing are described elsewhere [33]. Tissues were fixed in Fekete’s acid-alcohol-formalin solution. Sections were stained with hematoxylin and eosin (H&E) for routine screening.

Abbreviations

Far2: fatty acyl CoA reductase 2 gene
GLT: germ line transmitting
H&E: hematoxylin and eosin stain
MGI: Mouse Genome Informatics Database

Data Availability

All relevant data are within the paper.

Funding Statement

This work was supported by grants from the National Institutes of Health (R21-AR063781 and U42-OD011185). Shared services at The Jackson Laboratory are subsidized by a National Cancer Institute Core Grant (P30-CA034196). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All relevant data are within the paper.

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PERMALINK

Systematic screening for skin, hair, and nail abnormalities in a large-scale knockout mouse program

John P Sundberg

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Kathleen A Silva

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Stephen A Murray

Beth A Sundberg

Paul N Schofield