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Milk lacking α-casein leads to permanent reduction in body size in mice.

Kolb AF, Huber RC, Lillico SG, Carlisle A, Robinson CJ, Neil C, Petrie L, Sorensen DB, Olsson IA, Whitelaw CB - PLoS ONE (2011)

Bottom Line: In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced.The absence of α-casein also significantly inhibits transcription of the other casein genes. α-Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups.The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight.

View Article: PubMed Central - PubMed

Affiliation: Molecular Recognition Group, Hannah Research Institute, Ayr, United Kingdom. a.kolb@abdn.ac.uk

ABSTRACT
The major physiological function of milk is the transport of amino acids, carbohydrates, lipids and minerals to mammalian offspring. Caseins, the major milk proteins, are secreted in the form of a micelle consisting of protein and calcium-phosphate.We have analysed the role of the milk protein α-casein by inactivating the corresponding gene in mice. Absence of α-casein protein significantly curtails secretion of other milk proteins and calcium-phosphate, suggesting a role for α-casein in the establishment of casein micelles. In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced. The absence of α-casein also significantly inhibits transcription of the other casein genes. α-Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups. The data support a critical role for α-casein in casein micelle assembly. The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight.

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Targeting of the α-casein gene.Panel A: Schematic representation of the murine casein locus. Casein genes are represented as solid arrows. Other predicted genes are shown as open arrows. Panel B: Schematic representation of the unmodified α-casein gene and the targeted α-casein gene. Exons of the α-casein gene are indicated as solid boxes, the hytk selection marker gene is indicated as striped box. The PGK promoter element directing expression of the selection marker gene is indicated as arrowhead. The relative positions of the EcoRI restriction sites (EI), the Southern blot probe (probe), sizes of hybridising DNA fragments and the primer binding sites used for genotyping (horizontal arrows) are indicated. Panel C: PCR analysis of genomic DNA isolated from the three representative ES cell clones using the primer combination acas6, acas7 and pBKpA2 (analysing the 5′ end of the homologous recombination event). A 1566 bp band is detected in all samples and represents the unmodified α-casein allele [U: unmodified]. The second clone carries a targeted α-casein allele as indicated by the occurrence of a 848 bp PCR product [T: targeted]. Marker: phage λ digested with HindIII and EcoRI (λ×H/E). Panel D: Southern blot analysis of EcoRI digested DNA derived from tail clips of a wild-type (acas [+/+]), and α-casein mutant mice (heterozygous: acas [+/−], and homozygous α-casein [−/−]). The probe indicated in panel B detects a 7.5 kb DNA fragment representative of the unmodified α-casein allele [U] and a 4.3 kb band representative of the targeted α-casein allele [T]. Panel E: PCR analysis of genomic DNA isolated from two ES cell clones using the primer combination acas1, acas7 and PGK5 (analysing the 3′ end of the homologous recombination event). A 688 bp band is detected in both samples and represents the unmodified α-casein allele [U]. The second clone carries a targeted α-casein allele as indicated by the occurrence of a 450 bp PCR product [T]. Marker: NEB PCR marker.
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pone-0021775-g001: Targeting of the α-casein gene.Panel A: Schematic representation of the murine casein locus. Casein genes are represented as solid arrows. Other predicted genes are shown as open arrows. Panel B: Schematic representation of the unmodified α-casein gene and the targeted α-casein gene. Exons of the α-casein gene are indicated as solid boxes, the hytk selection marker gene is indicated as striped box. The PGK promoter element directing expression of the selection marker gene is indicated as arrowhead. The relative positions of the EcoRI restriction sites (EI), the Southern blot probe (probe), sizes of hybridising DNA fragments and the primer binding sites used for genotyping (horizontal arrows) are indicated. Panel C: PCR analysis of genomic DNA isolated from the three representative ES cell clones using the primer combination acas6, acas7 and pBKpA2 (analysing the 5′ end of the homologous recombination event). A 1566 bp band is detected in all samples and represents the unmodified α-casein allele [U: unmodified]. The second clone carries a targeted α-casein allele as indicated by the occurrence of a 848 bp PCR product [T: targeted]. Marker: phage λ digested with HindIII and EcoRI (λ×H/E). Panel D: Southern blot analysis of EcoRI digested DNA derived from tail clips of a wild-type (acas [+/+]), and α-casein mutant mice (heterozygous: acas [+/−], and homozygous α-casein [−/−]). The probe indicated in panel B detects a 7.5 kb DNA fragment representative of the unmodified α-casein allele [U] and a 4.3 kb band representative of the targeted α-casein allele [T]. Panel E: PCR analysis of genomic DNA isolated from two ES cell clones using the primer combination acas1, acas7 and PGK5 (analysing the 3′ end of the homologous recombination event). A 688 bp band is detected in both samples and represents the unmodified α-casein allele [U]. The second clone carries a targeted α-casein allele as indicated by the occurrence of a 450 bp PCR product [T]. Marker: NEB PCR marker.

Mentions: In order to inactivate the α-casein gene (Fig. 1a) a targeting construct was established based on previous experience with constructs used to inactivate the β- and γ-casein genes [5], [27]. The targeting event removes the complete second exon which includes the translational start codon and the 15 amino acid signal peptide (Fig. 1b). In 3 independent transfections into ES cells the targeting frequency was found to be around 3%. Successfully modified ES cells clones were identified using a PCR analysing the 5′ end of the targeted gene (Fig. 1c). The targeting event was then confirmed by Southern blot analysis assessing the 5′ end of the targeted α-casein gene (Fig. 1d). The integration event was also confirmed using PCR analysis and Southern blotting with primer combinations and probes specific for the 3′ end (Fig. 1e). Two targeted cell clones were grown up and injected into blastocysts. Animals carrying a homozygous deletion of the α-casein gene are phenotypically normal prior to lactation.


Milk lacking α-casein leads to permanent reduction in body size in mice.

Kolb AF, Huber RC, Lillico SG, Carlisle A, Robinson CJ, Neil C, Petrie L, Sorensen DB, Olsson IA, Whitelaw CB - PLoS ONE (2011)

Targeting of the α-casein gene.Panel A: Schematic representation of the murine casein locus. Casein genes are represented as solid arrows. Other predicted genes are shown as open arrows. Panel B: Schematic representation of the unmodified α-casein gene and the targeted α-casein gene. Exons of the α-casein gene are indicated as solid boxes, the hytk selection marker gene is indicated as striped box. The PGK promoter element directing expression of the selection marker gene is indicated as arrowhead. The relative positions of the EcoRI restriction sites (EI), the Southern blot probe (probe), sizes of hybridising DNA fragments and the primer binding sites used for genotyping (horizontal arrows) are indicated. Panel C: PCR analysis of genomic DNA isolated from the three representative ES cell clones using the primer combination acas6, acas7 and pBKpA2 (analysing the 5′ end of the homologous recombination event). A 1566 bp band is detected in all samples and represents the unmodified α-casein allele [U: unmodified]. The second clone carries a targeted α-casein allele as indicated by the occurrence of a 848 bp PCR product [T: targeted]. Marker: phage λ digested with HindIII and EcoRI (λ×H/E). Panel D: Southern blot analysis of EcoRI digested DNA derived from tail clips of a wild-type (acas [+/+]), and α-casein mutant mice (heterozygous: acas [+/−], and homozygous α-casein [−/−]). The probe indicated in panel B detects a 7.5 kb DNA fragment representative of the unmodified α-casein allele [U] and a 4.3 kb band representative of the targeted α-casein allele [T]. Panel E: PCR analysis of genomic DNA isolated from two ES cell clones using the primer combination acas1, acas7 and PGK5 (analysing the 3′ end of the homologous recombination event). A 688 bp band is detected in both samples and represents the unmodified α-casein allele [U]. The second clone carries a targeted α-casein allele as indicated by the occurrence of a 450 bp PCR product [T]. Marker: NEB PCR marker.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3138747&req=5

pone-0021775-g001: Targeting of the α-casein gene.Panel A: Schematic representation of the murine casein locus. Casein genes are represented as solid arrows. Other predicted genes are shown as open arrows. Panel B: Schematic representation of the unmodified α-casein gene and the targeted α-casein gene. Exons of the α-casein gene are indicated as solid boxes, the hytk selection marker gene is indicated as striped box. The PGK promoter element directing expression of the selection marker gene is indicated as arrowhead. The relative positions of the EcoRI restriction sites (EI), the Southern blot probe (probe), sizes of hybridising DNA fragments and the primer binding sites used for genotyping (horizontal arrows) are indicated. Panel C: PCR analysis of genomic DNA isolated from the three representative ES cell clones using the primer combination acas6, acas7 and pBKpA2 (analysing the 5′ end of the homologous recombination event). A 1566 bp band is detected in all samples and represents the unmodified α-casein allele [U: unmodified]. The second clone carries a targeted α-casein allele as indicated by the occurrence of a 848 bp PCR product [T: targeted]. Marker: phage λ digested with HindIII and EcoRI (λ×H/E). Panel D: Southern blot analysis of EcoRI digested DNA derived from tail clips of a wild-type (acas [+/+]), and α-casein mutant mice (heterozygous: acas [+/−], and homozygous α-casein [−/−]). The probe indicated in panel B detects a 7.5 kb DNA fragment representative of the unmodified α-casein allele [U] and a 4.3 kb band representative of the targeted α-casein allele [T]. Panel E: PCR analysis of genomic DNA isolated from two ES cell clones using the primer combination acas1, acas7 and PGK5 (analysing the 3′ end of the homologous recombination event). A 688 bp band is detected in both samples and represents the unmodified α-casein allele [U]. The second clone carries a targeted α-casein allele as indicated by the occurrence of a 450 bp PCR product [T]. Marker: NEB PCR marker.
Mentions: In order to inactivate the α-casein gene (Fig. 1a) a targeting construct was established based on previous experience with constructs used to inactivate the β- and γ-casein genes [5], [27]. The targeting event removes the complete second exon which includes the translational start codon and the 15 amino acid signal peptide (Fig. 1b). In 3 independent transfections into ES cells the targeting frequency was found to be around 3%. Successfully modified ES cells clones were identified using a PCR analysing the 5′ end of the targeted gene (Fig. 1c). The targeting event was then confirmed by Southern blot analysis assessing the 5′ end of the targeted α-casein gene (Fig. 1d). The integration event was also confirmed using PCR analysis and Southern blotting with primer combinations and probes specific for the 3′ end (Fig. 1e). Two targeted cell clones were grown up and injected into blastocysts. Animals carrying a homozygous deletion of the α-casein gene are phenotypically normal prior to lactation.

Bottom Line: In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced.The absence of α-casein also significantly inhibits transcription of the other casein genes. α-Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups.The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight.

View Article: PubMed Central - PubMed

Affiliation: Molecular Recognition Group, Hannah Research Institute, Ayr, United Kingdom. a.kolb@abdn.ac.uk

ABSTRACT
The major physiological function of milk is the transport of amino acids, carbohydrates, lipids and minerals to mammalian offspring. Caseins, the major milk proteins, are secreted in the form of a micelle consisting of protein and calcium-phosphate.We have analysed the role of the milk protein α-casein by inactivating the corresponding gene in mice. Absence of α-casein protein significantly curtails secretion of other milk proteins and calcium-phosphate, suggesting a role for α-casein in the establishment of casein micelles. In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced. The absence of α-casein also significantly inhibits transcription of the other casein genes. α-Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups. The data support a critical role for α-casein in casein micelle assembly. The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight.

Show MeSH
Related in: MedlinePlus