A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains

Background

The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.

Results

We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.

Conclusions

Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.     

Next-generation sequencing of experimental mouse strains

Since the turn of the century the complete genome sequence of just one mouse strain, C57BL/6J, has been available. Knowing the sequence of this strain has enabled large-scale forward genetic screens to be performed, the creation of an almost complete set of embryonic stem (ES) cell lines with targeted alleles for protein-coding genes, and the generation of a rich catalog of mouse genomic variation. However, many experiments that use other common laboratory mouse strains have been hindered by a lack of whole-genome sequence data for these strains. The last 5?years has witnessed a revolution in DNA sequencing technologies. Recently, these technologies have been used to expand the repertoire of fully sequenced mouse genomes. In this article we review the main findings of these studies and discuss how the sequence of mouse genomes is helping pave the way from sequence to phenotype. Finally, we discuss the prospects for using de novo assembly techniques to obtain high-quality assembled genome sequences of these laboratory mouse strains, and what advances in sequencing technologies may be required to achieve this goal.     

Molecular evolution of <Emphasis Type="Italic">V</Emphasis><Subscript><Emphasis Type="Italic">H</Emphasis></Subscript><Emphasis Type="Italic">9</Emphasis> germline genes isolated from DBA,BALB, 129 and C57BL mouse strains and sublines

We have used the polymerase chain reaction (PCR) in an attempt to clone and sequence the exons and hitherto unavailable contiguous flanks of all members of the small V(H) 9 germline gene family from inbred mouse strains and sublines that have had a common ancestry within the last century, and to analyze the molecular evolution of these sequences. Fifteen genuine germline genes were isolated (designated V(H) 9.1 through V(H) 9.15) from strains and sublines of DBA, BALB, 129 and C57BL inbred mice. Of the 15 genuine isolates, nine are novel: seven sequences from DBA strains and sublines ( V(H) 9.3 to V(H) 9.9) and two sequences from C57BL strains ( V(H) 9.13 and V(H) 9.14). We have identified sequencing errors and PCR recombinant artefacts in previously published sequences. We detected no sequence divergence of individual genes shared by the strains and sublines studied. However, we isolated two genes from DBA strains and sublines, V(H) 9.1 and V(H) 9.3, that differ only by five nucleotides encoding three amino acid changes that are concentrated within a 33 nucleotide (11 codon) region. Of these 11 codons, eight encode a putative antigen binding site. There were no differences in the remaining 733 nucleotides sequenced (including both 5' and 3' flanking regions). Potential explanations for the generation of V(H) 9.1 and V(H) 9.3 are discussed.     

Eye lens proteomics: from global approach to detailed information about phakinin and gamma E and F crystallin genes

Exploration of the lenticular proteome poses a challenging and worthwhile undertaking as cataracts, the products of a disease phenotype elicited by this proteome, remains the leading cause of vision impairment worldwide. The complete ten day old lens proteome of Mus musculus C57BL/6J was resolved into 900 distinct spots by large gel carrier ampholyte based 2-DE. The predicted amino acid sequences of all 16 crystallins ubiquitous in mammals were corroborated by mass spectrometry (MS). In detailed individual spot analyses, the primary structure of the full murine C57BL/6J beaded filament component phakinin CP49 was sequenced by liquid chromatography/electrospray ionization-tandem MS and amended at two positions. This definitive polypeptide sequence was aligned to the mouse genome, thus identifying the entire C57BL/6J genomic coding region. Also, two murine C57/6J polypeptides, both previously classified as gamma F crystallin, were clearly distinguished by MS and electrophoretic mobility. Both were assigned to their respective genes, one of the polypeptides was reclassified as C57BL/6J gamma E crystallin. Building on these data and previous investigations an updated crystallin reference map was put forth and several non crystallin lenticular components were examined. These results represent the first part of a comprehensive investigation of the mouse lens proteome (http://www.mpiib-berlin.mpg.de/2D-PAGE) with emphasis on understanding genetic effects on proteins and disease development.     

Two induced anti-Z-DNA monoclonal antibodies use VH gene segments related to those of anti-DNA autoantibodies

The cDNA for H and L chain V regions of two anti-Z-DNA mAb, Z22 and Z44, were cloned and sequenced. These are the first experimentally induced anti-nucleic acid antibody sequences available for comparison with autoantibody sequences. Z22 and Z44 are IgG2b and IgG2a antibodies from C57BL/6 mice. They recognize different facets of the Z-DNA structure. They both use VH10 family genes and share 95% sequence base sequence identity in the VH and leader sequences; however, they differ in the 5'-untranslated region of the VH mRNA, indicating they arise from different germline genes. Both use JH4 segments. They differ from each other very extensively in the CDR3 of both H and L chains. The most closely related H chains in the current GenBank/EMBL data base are two mouse IgG anti-DNA autoantibodies, one from an MRL-lpr/lpr mouse (MRL-DNA4) and one from an NZB/NZW mouse (BV04-01). Z22 and Z44 share 95% sequence identity with these antibodies in the VH segment. In addition, Z22 is identical to MRL-DNA4 at 91% of the positions in the 5'-untranslated region of the H chain mRNA. The two antibodies share 95% base sequence identity in the V kappa segment. The most closely related L chains, with 97 to 98% sequence identity, are the V kappa 10b germline gene for Z22 and the V kappa 10a germ line gene, which is associated with A/J anti-arsonate antibodies and BALB/c anti-ABO blood group substance antibodies, for Z44. Z22 and Z44 share several structural features (similarities in VH, JH, and V kappa) but differ very markedly in the L chain CDR1 and both H and L chain CDR3 sequences; these regions may determine the differences in their specific interactions with Z-DNA.     

Identification of 129S6/SvEvTac-specific polymorphisms on mouse chromosome 11

Polymorphisms such as single-nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) can be associated with phenotypic traits and be used as markers for disease diagnosis. Identification of these genetic variations within laboratory mice is crucial to improve our understanding of the genetic background of the mice used for research. As part of a positional cloning project, we sequenced six genes (Mettl16, Evi2a, Psmd11, Cct6d, Rffl, and Ap2b1) within a 6.8-Mb domain of mmu chr 11 in the C57BL/6J and 129S6/SvEvTac inbred strains. Although 129S6/SvEvTac is widely used in the mouse community, there is very little current (or projected future) sequence information available for this strain. We identified 6 Indels and 21 novel SNPs and confirmed genotype information for 114 additional SNPs in these 6 genes. Mettl16 and Ap2b1 contained the largest numbers of variants between the C57BL/6J and 129S6/SvEvTac strains. In addition, we found five new SNPs between 129S6/SvEvTac and 129S1/SvImJ within the Ap2b1 locus. Although we did not detect differences between C57BL/6J and 129S6/SvEvTac within Evi2a, this locus contains a relatively high SNP density compared with the surrounding sequence. Our study highlights the genetic differences among three inbred mouse strains (C57BL/6J, 129S6/SvEvTac, and 129S1/SvImJ) and provides valuable sequence information that can be used to track alleles in genomics-based studies.     

Maternal modulation of the inheritable meiosis I error Dipl I in mouse oocytes is associated with the type of mitochondrial DNA

Summary The ovulation of diploid oocytes, abnormally arrested at or during the first meiotic division, is an inheritable trait (Dipl I) in mice and modulated by a maternally transmitted factor. By repeated backcrossing, mouse strains with identical nuclear encoded genes and differing only in their mitochondrial genomes can be created. NMB mice represent such a strain having acquired the nuclear genome of C57BL/6J but still possessing mitochondria and therewith mitochondrial DNA (mtDNA) of NMRI/Bom, their female progenitor. The strains NMB and C57BL/6J were used to characterize a new mitochondrial trait, namely the ability to modulate the expression of the inheritable meiosis I error Dipl I in oocytes. We show that an increased rate of ovulated diploid oocytes is associated with the mtDNA type of C57BL/6J. These results corroborate the assumption that mitochondria do play an important role in meiosis of mammalian oocytes and hence seem to be involved also in the orderly segregation of chromosomes.     

Sequences of mouse immunoglobulin light chain genes before and after somatic changes.

We have determined the nucleotide sequences of the germ line gene as well as a corresponding somatically mutated and rearranged gene coding for a mouse immunoglobulin lambdaI type light chain. These sequencing studies were carried out on three Eco RI-DNA fragments which had been cloned from BALB/c mouse embryos or a lambdaI chainsecreting myeloma, H2020. The embryonic DNA clone Ig 99lambda contains two protein-encoding segments, one for the majority of the hydrophobic leader (L) and the other for the rest of the leader and the variable (V) region of the lambda0 chain (Cohn et al., 1974); these segments are separated by a 93 base pair (bp) intervening sequence (I-small). The coding of the V region ends with His at residue 97. The second embryonic DNA clone Ig 25lambda includes a 39 bp DNA segment (J) coding for the rest of the conventionally defined V region (that is, up to residue 110), and also contains the sequence coding for the constant (C) region approximately 1250 untranslated bp (I-large) away from the J sequence. The J sequence is directly linked with the V-coding sequence in the myeloma DNA clone, Ig 303lambda, which has the various DNA segments arranged in the following order: 5' untranslated region, L, l-small, V linked with J, l-large, C, 3' untranslated sequence. The lg 303lambda V DNA sequence codes for the V region synthesized by the H2020 myeloma and is different from the lg 99lambda V DNA sequence by only two bases. No silent base change was observed between the two DNA clones for the entire sequence spanning the 5' untranslated regions and the V-coding segments. These results confirm the previously drawn conclusion that an active complete lambdaI gene arises by somatic recombination that takes place at the ends of the V-coding DNA segment and the J sequence. No sequence homology was observed at or near the sites of the recombination.     

Identification and characterization of functional genes encoding the mouse major urinary proteins.

Mouse Ltk- cells were stably transfected with cloned genes encoding the mouse major urinary proteins (MUPs). C57BL/6J MUP genomic clones encoding MUP 2 (BL6-25 and BL6-51), MUP 3 (BL6-11 and BL6-3), and MUP 4 (BL6-42) have been identified. In C57BL/6J mice, MUP 2 and MUP 4 are known to be synthesized in male, but not female, liver, and MUP 3 is known to be synthesized in both male and female liver and mammary gland. A BALB/c genomic clone (BJ-31) was shown to encode a MUP that is slightly more basic than MUP 2 and was previously shown to be synthesized in both male and female liver of BALB/c but not C57BL/6 mice. Comigration on two-dimensional polyacrylamide gels of the MUPs encoded by the transfecting gene provides a basis for tentative identification of the tissue specificity and mode of regulation of each gene. DNA sequence analysis of the 5' flanking region indicates that the different MUP genes are highly homologous (0.20 to 2.40% divergence) within the 879 base pairs analyzed. The most prominent differences in sequence occur within an A-rich region just 5' of the TATA box. This region (from -47 to -93) contains primarily A or C(A)N nucleotides and varies from 15 to 46 nucleotides in length in the different clones.     

Reconstructing the plant mitochondrial genome for marker discovery: a case study using Pinus

Whole‐genome‐shotgun (WGS) sequencing of total genomic DNA was used to recover ~1 Mbp of novel mitochondrial (mtDNA) sequence from Pinus sylvestris (L.) and three members of the closely related Pinus mugo species complex. DNA was extracted from megagametophyte tissue from six mother trees from locations across Europe, and 100‐bp paired‐end sequencing was performed on the Illumina HiSeq platform. Candidate mtDNA sequences were identified by their size and coverage characteristics, and by comparison with published plant mitochondrial genomes. Novel variants were identified, and primers targeting these loci were trialled on a set of 28 individuals from across Europe. In total, 31 SNP loci were successfully resequenced, characterizing 15 unique haplotypes. This approach offers a cost‐effective means of developing marker resources for mitochondrial genomes in other plant species where reference sequences are unavailable.     

A nonpolymorphic class I gene in the murine major histocompatibility complex

DNA sequence analysis of a class I gene (Q10), which maps to the Qa2,3 locus in the C57BL/10 (H-2b haplotype) mouse, reveals that it is almost identical to a cDNA clone (pH16) isolated from a SWR/J (H-2q haplotype) mouse liver cDNA library. Exon 5, in particular, has an unusual structure such that a polypeptide product is unlikely to be anchored in the cell membrane. Our findings suggest that the two sequences are derived from allelic class I genes, which are nonpolymorphic, in contrast to H-2K allelic sequences from the same mice, and they may encode liver-specific polypeptides of unknown function. Our previous studies indicate that the Q10 gene is a potential donor gene for the generation of mutations at the H-2K locus by inter-gene transfer of genetic information. Thus the lack of polymorphism in class I genes at the Q10 locus implies either that they are not recipients for such exchanges or that selective pressure prevents the accumulation of mutations in genes at this locus.     

Construction of a BAC Contig for a 3 cM Biologically Significant Region of Mouse Chromosome 1

One QTL and genes and phenotypes have been localized in the region between 92 cM and 95cM of mouse chromosome 1. The QTL locus contributes to approximately 40% of the variation of the peak bone density between C57BL/6J (B6) and CAST/EiJ (CAST) strains. Other loci located in this chromosomal region include a neural tube defect mutant loop-tail (Lp), a lymphocyte-stimulating determinant (Lsd), and the Transgelin 2 (Tagln 2). The human chromosome region homologous to this region is 1q21-23, which also contains a QTL locus for high bone mineral density (BMD). Furthermore, it has been reported that this region may have duplicated several times in the mouse genome. Therefore, genomic sequencing of this region will provide important information for mouse genome structure, for positional cloning of mouse genes, and for the study of human homologous genes. In order to provide a suitable template for genomic sequencing by the NIH-sponsored genomic centers, we have constructed a BAC contig of this region using the RPCI-23 library. We have also identified the currently available mouse genomic sequences localized in our BAC contig. Further analysis of these sequences and BAC clones indicated a high frequency of repetitive sequences within this chromosomal area. This region also contains L1 retrotransposon sequences, providing a potential mechanism for the repetitive sequences described in the literature.     

Normal variants of human mitochondrial DNA and translation products: the building of a reference data base

Summary A good standard reference for the highly polymorphic human mitochondrial DNA (mtDNA) sequence is essential for studies of normal and disease-related nucleotide variants in the mitochondrial genome. A consensus sequence for the human mitochondrial genome has been derived from thirteen unrelated mtDNA sequences. We report 128 nucleotide variants of the human mtDNA sequence, and 62 amino acid variants of the human mitochondrial translation products, observed in the coding region of these mtDNA sequences.     

Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences.

Two functional cytosolic thymidine kinase (tk) cDNA clones were isolated from a mouse L-cell library. An RNA blot analysis indicated that one of these clones contains a nearly full-length tk sequence and that LTK- cells contain little or no TK message. The nucleotide sequences of both clones were determined, and the functional mouse tk cDNA contains 1,156 base pairs. An analysis of the sequence implied that there is an untranslated 32-nucleotide region at the 5' end of the mRNA, followed by an open reading frame of 699 nucleotides. The 3' untranslated region is 422 nucleotides long. Thus, the gene codes for a protein containing 233 amino acids, with a molecular weight of 25,873. A comparison of the coding sequences of the mouse tk cDNA with the human and chicken tk genes revealed about 86 and 70% homology, respectively. We also isolated the tk gene from a mouse C57BL/10J cosmid library. The structural organization was determined by restriction mapping, Southern blotting, and heteroduplex analysis of the cloned sequences, in combination with a mouse tk cDNA. The tk gene spans approximately 11 kilobases and contains at least five introns. Southern blot analysis revealed that this gene is deleted in mouse LTK- cells, consistent with the inability of these cells to synthesize TK message. This analysis also showed that tk-related sequences are present in the genomes of several mouse strains, as well as in LTK- cells. These segments may represent pseudogenes.     

Evaluating gene expression in C57BL/6J and DBA/2J mouse striatum using RNA-Seq and microarrays

C57BL/6J (B6) and DBA/2J (D2) are two of the most commonly used inbred mouse strains in neuroscience research. However, the only currently available mouse genome is based entirely on the B6 strain sequence. Subsequently, oligonucleotide microarray probes are based solely on this B6 reference sequence, making their application for gene expression profiling comparisons across mouse strains dubious due to their allelic sequence differences, including single nucleotide polymorphisms (SNPs). The emergence of next-generation sequencing (NGS) and the RNA-Seq application provides a clear alternative to oligonucleotide arrays for detecting differential gene expression without the problems inherent to hybridization-based technologies. Using RNA-Seq, an average of 22 million short sequencing reads were generated per sample for 21 samples (10 B6 and 11 D2), and these reads were aligned to the mouse reference genome, allowing 16,183 Ensembl genes to be queried in striatum for both strains. To determine differential expression, 'digital mRNA counting' is applied based on reads that map to exons. The current study compares RNA-Seq (Illumina GA IIx) with two microarray platforms (Illumina MouseRef-8 v2.0 and Affymetrix MOE 430 2.0) to detect differential striatal gene expression between the B6 and D2 inbred mouse strains. We show that by using stringent data processing requirements differential expression as determined by RNA-Seq is concordant with both the Affymetrix and Illumina platforms in more instances than it is concordant with only a single platform, and that instances of discordance with respect to direction of fold change were rare. Finally, we show that additional information is gained from RNA-Seq compared to hybridization-based techniques as RNA-Seq detects more genes than either microarray platform. The majority of genes differentially expressed in RNA-Seq were only detected as present in RNA-Seq, which is important for studies with smaller effect sizes where the sensitivity of hybridization-based techniques could bias interpretation.     

Structural evidence for a polymorphic or allelic form of the heavy chain variable region.

The heavy (H) chains of anti-phosphocholine (PC) antibodies from C57BL/6J and CBA/J were sequenced through the N-terminal 36 residues and compared with previously published sequences of A/J anti-PC antibody and BALB/c PC-binding myeloma proteins T15, M603, and M511. Each of these antibody preparations contained molecules having light (L) chains and idiotypic determinants of T15, M511, and M603 indicating the presence of at least three different anti-PC antibodies in each pool. The structures of the C57BL/6J and CBA/J H chains each revealed a single sequence from positions 1 to 36 (which includes the first complementarity determining region (CDR), and they were identical. The first CDR was identical to that previously found for BALB/c and A/J indicating that this portion of these antibody molecules is highly conserved throughout inbred mice and is probably critical to PC-binding. A surprising finding was that both C57NL/6 and CBA sequence differed from the BALB/c and A/J sequences at two positions, residue 14 and 16. Since each of these strains differs at the allotype locus, the data indicates that the evolution of allotypy in mice occurred after variable region diversity for the particular genes.     

DNA sequence error rates in Genbank records estimated using the mouse genome as a reference.

We estimate DNA sequence error rates in Genbank records containing protein-coding and non-coding DNA sequences by comparing sequences of the inbred mouse strain C57BL/6J, sequenced as part of the mouse genome project and independently by other laboratories. C57BL/6J was produced by more than 100 generations of brother-sister mating, and can be assumed to be virtually free of residual polymorphism and mutational variation, so differences between independent sequences can be attributed to error. The estimated single nucleotide error rate for coding DNA is 0.10% (SE 0.012%), which is substantially lower than previous estimates for error rates in Genbank accessions. The estimated single nucleotide error rate for intronic DNA sequences (0.22%; SE 0.051%) is significantly higher than the rate for coding DNA. Since error rates for the mouse genome sequence are very low, the vast majority of the errors we detected are likely to be in individual Genbank accessions. The frequency of insertion-deletion (indel) errors in non-coding DNA approaches that of single nucleotide errors in non-coding DNA, whereas indel errors are uncommon in coding sequences.     

Subfamilies of the mouse major urinary protein (MUP) multi-gene family: sequence analysis of cDNA clones and differential regulation in the liver

The mouse major urinary proteins (MUPs) are the products of a multi-gene family of 30-35 genes whose members exhibit diverse tissue specific, developmental, and hormonal controls. Three cDNA clones corresponding to liver MUP mRNAs have been sequenced. Two of the clones (p499, C57BL/6 and p1057, BALB/c) share strong homology whereas a third clone (p199, C57BL/6) has diverged considerably from the others at the nucleic acid (85% homology) and protein (68% homology) levels. The 5' regions of p499 and p199 which show the most sequence divergence were subcloned and shown to hybridize to different liver MUP mRNAs. The p499-5' sequence was expressed in all MUP expressing tissues (liver, lachrymal, submaxillary and mammary) whereas the p199-5' sequence was expressed primarily in the liver and lachrymal. Analysis of liver RNA from mice in different endocrine states indicates that the p499-5' sequence is strongly regulated by thyroxine administration whereas the p199-5' sequence is not. Both sequences appear to be regulated by growth hormone and testosterone. Southern blot analysis of mouse genomic DNA indicates that there are multiple genes homologous to each sequence.