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Segregation of a spontaneous Klrd1 (CD94) mutation in DBA/2 mouse substrains.

Shin DL, Pandey AK, Ziebarth JD, Mulligan MK, Williams RW, Geffers R, Hatesuer B, Schughart K, Wilk E - G3 (Bethesda) (2014)

Bottom Line: For example, BXD lines (BXD1-32) generated in the 1970s by crossing B6 and D2J do not segregate for the exonic deletion and have high expression, whereas BXD lines 33 and greater were generated after 1990 are segregating for the deletion and have highly variable Klrd1 expression.We performed quantitative trait locus analysis of Klrd1 expression by using BXD lines with different generation times and found that the expression difference in Klrd1 in the later BXD set is driven by a strong cis-acting expression quantitative trait locus.Although the Klrd1/CD94 locus is essential for mousepox resistance, the genetic variation among D2 substrains and the later set of BXD strains is not associated with susceptibility to the Influenza A virus PR8 strain.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection Genetics, Helmholtz Centre for Infection Research and University of Veterinary Medicine Hannover, Braunschweig, Germany.

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Characterization of the deletions in Klrd1 gene in D2J and D2Rj. (A) Schematic representation of transcribed region at the Klrd1 locus. Two deletions were observed in the Klrd1 gene of D2J mice. The first deletion is located in intron 2, the second deletion starts in intron 4 and contains the entire exon 5. Both D2 strains carry the first deletion, whereas only D2J carries the downstream deletion that encodes a nonfunctional truncated gene product. In addition, sequence analysis of the BXD29 genome confirmed the structure of the “old” D2 allele that is carried by D2Rj mice: a deletion in intron 2 but not in exon 5. (B) Polymerase chain reaction (PCR) performed with genomic DNA from B6, D2J, and D2Rj mice detected the deletion in intron 2. DNA from both D2 strains generated a PCR product of 158 bp in length and B6 of 349 bp, respectively. Primers were as follows: fw-5′atacatgyttcctaacgagtgttc and rev-5′aaggtctattcttatagagatgtctatact. (C) In intron 2, a 191-bp deletion was observed in both D2 strains and a tandem repeat in B6 (underlined). Sequence alignment was performed with Martinez/Needleman-Wunsch method by MegAlign (DNASTAR, USA).
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fig3: Characterization of the deletions in Klrd1 gene in D2J and D2Rj. (A) Schematic representation of transcribed region at the Klrd1 locus. Two deletions were observed in the Klrd1 gene of D2J mice. The first deletion is located in intron 2, the second deletion starts in intron 4 and contains the entire exon 5. Both D2 strains carry the first deletion, whereas only D2J carries the downstream deletion that encodes a nonfunctional truncated gene product. In addition, sequence analysis of the BXD29 genome confirmed the structure of the “old” D2 allele that is carried by D2Rj mice: a deletion in intron 2 but not in exon 5. (B) Polymerase chain reaction (PCR) performed with genomic DNA from B6, D2J, and D2Rj mice detected the deletion in intron 2. DNA from both D2 strains generated a PCR product of 158 bp in length and B6 of 349 bp, respectively. Primers were as follows: fw-5′atacatgyttcctaacgagtgttc and rev-5′aaggtctattcttatagagatgtctatact. (C) In intron 2, a 191-bp deletion was observed in both D2 strains and a tandem repeat in B6 (underlined). Sequence alignment was performed with Martinez/Needleman-Wunsch method by MegAlign (DNASTAR, USA).

Mentions: To further investigate the basis of the difference in expression of CD94 in these D2 substrains, we analyzed high-throughput sequence data for D2J (~100X) and low coverage sequence data for D2Rj (5.5X). We confirmed the complete deletion of the last exon (exon 5) and 3′ UTR and a partial deletion of intron 4 of Klrd1 in D2J (Figure 3B) using next-generation sequencing based on different structural variant detection approaches (Supporting Information, File S1). The discordant mate-pair approach detected a deletion on chr6 from 129,597,284 to 129,600,182 bp (mm10 assembly) and the read-depth approach detected a similarly sized deletion from 129,597,400 to 129,599,450 bp (mm10 assembly). However, we could not locate this deletion in D2Rj by using next-generation sequencing data. In addition, we performed polymerase chain reaction (PCR) analysis by using primers specific to exon 5—the key deleted region. No PCR product was detected in D2J, but we were able to amplify this exon in D2Rj (Figure 2C and Figure S1A). The sequence of the product showed 98% sequence identity with B6. The deletion in D2J starts within intron 5 (Figure 3A), consistent with previous findings (Wilhelm et al. 2003). In addition, both substrains contain a 191-bp deletion in the intronic region between exons 2 and 3. This second deletion was characterized by PCR (Figure 3, B and C and Figure S1B); D2J chr6:129,594,469-129,594,667, D2Rj chr6:129,594,271-129,594,389, mm10 assembly).


Segregation of a spontaneous Klrd1 (CD94) mutation in DBA/2 mouse substrains.

Shin DL, Pandey AK, Ziebarth JD, Mulligan MK, Williams RW, Geffers R, Hatesuer B, Schughart K, Wilk E - G3 (Bethesda) (2014)

Characterization of the deletions in Klrd1 gene in D2J and D2Rj. (A) Schematic representation of transcribed region at the Klrd1 locus. Two deletions were observed in the Klrd1 gene of D2J mice. The first deletion is located in intron 2, the second deletion starts in intron 4 and contains the entire exon 5. Both D2 strains carry the first deletion, whereas only D2J carries the downstream deletion that encodes a nonfunctional truncated gene product. In addition, sequence analysis of the BXD29 genome confirmed the structure of the “old” D2 allele that is carried by D2Rj mice: a deletion in intron 2 but not in exon 5. (B) Polymerase chain reaction (PCR) performed with genomic DNA from B6, D2J, and D2Rj mice detected the deletion in intron 2. DNA from both D2 strains generated a PCR product of 158 bp in length and B6 of 349 bp, respectively. Primers were as follows: fw-5′atacatgyttcctaacgagtgttc and rev-5′aaggtctattcttatagagatgtctatact. (C) In intron 2, a 191-bp deletion was observed in both D2 strains and a tandem repeat in B6 (underlined). Sequence alignment was performed with Martinez/Needleman-Wunsch method by MegAlign (DNASTAR, USA).
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Related In: Results  -  Collection

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fig3: Characterization of the deletions in Klrd1 gene in D2J and D2Rj. (A) Schematic representation of transcribed region at the Klrd1 locus. Two deletions were observed in the Klrd1 gene of D2J mice. The first deletion is located in intron 2, the second deletion starts in intron 4 and contains the entire exon 5. Both D2 strains carry the first deletion, whereas only D2J carries the downstream deletion that encodes a nonfunctional truncated gene product. In addition, sequence analysis of the BXD29 genome confirmed the structure of the “old” D2 allele that is carried by D2Rj mice: a deletion in intron 2 but not in exon 5. (B) Polymerase chain reaction (PCR) performed with genomic DNA from B6, D2J, and D2Rj mice detected the deletion in intron 2. DNA from both D2 strains generated a PCR product of 158 bp in length and B6 of 349 bp, respectively. Primers were as follows: fw-5′atacatgyttcctaacgagtgttc and rev-5′aaggtctattcttatagagatgtctatact. (C) In intron 2, a 191-bp deletion was observed in both D2 strains and a tandem repeat in B6 (underlined). Sequence alignment was performed with Martinez/Needleman-Wunsch method by MegAlign (DNASTAR, USA).
Mentions: To further investigate the basis of the difference in expression of CD94 in these D2 substrains, we analyzed high-throughput sequence data for D2J (~100X) and low coverage sequence data for D2Rj (5.5X). We confirmed the complete deletion of the last exon (exon 5) and 3′ UTR and a partial deletion of intron 4 of Klrd1 in D2J (Figure 3B) using next-generation sequencing based on different structural variant detection approaches (Supporting Information, File S1). The discordant mate-pair approach detected a deletion on chr6 from 129,597,284 to 129,600,182 bp (mm10 assembly) and the read-depth approach detected a similarly sized deletion from 129,597,400 to 129,599,450 bp (mm10 assembly). However, we could not locate this deletion in D2Rj by using next-generation sequencing data. In addition, we performed polymerase chain reaction (PCR) analysis by using primers specific to exon 5—the key deleted region. No PCR product was detected in D2J, but we were able to amplify this exon in D2Rj (Figure 2C and Figure S1A). The sequence of the product showed 98% sequence identity with B6. The deletion in D2J starts within intron 5 (Figure 3A), consistent with previous findings (Wilhelm et al. 2003). In addition, both substrains contain a 191-bp deletion in the intronic region between exons 2 and 3. This second deletion was characterized by PCR (Figure 3, B and C and Figure S1B); D2J chr6:129,594,469-129,594,667, D2Rj chr6:129,594,271-129,594,389, mm10 assembly).

Bottom Line: For example, BXD lines (BXD1-32) generated in the 1970s by crossing B6 and D2J do not segregate for the exonic deletion and have high expression, whereas BXD lines 33 and greater were generated after 1990 are segregating for the deletion and have highly variable Klrd1 expression.We performed quantitative trait locus analysis of Klrd1 expression by using BXD lines with different generation times and found that the expression difference in Klrd1 in the later BXD set is driven by a strong cis-acting expression quantitative trait locus.Although the Klrd1/CD94 locus is essential for mousepox resistance, the genetic variation among D2 substrains and the later set of BXD strains is not associated with susceptibility to the Influenza A virus PR8 strain.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection Genetics, Helmholtz Centre for Infection Research and University of Veterinary Medicine Hannover, Braunschweig, Germany.

Show MeSH
Related in: MedlinePlus