Limits...
A tubulin alpha 8 mouse knockout model indicates a likely role in spermatogenesis but not in brain development

View Article: PubMed Central - PubMed

ABSTRACT

Tubulin alpha 8 (Tuba8) is the most divergent member of the highly conserved alpha tubulin family, and uniquely lacks two key post-translational modification sites. It is abundantly expressed in testis and muscle, with lower levels in the brain. We previously identified homozygous hypomorphic TUBA8 mutations in human subjects with a polymicrogyria (PMG) syndrome, suggesting its involvement in development of the cerebral cortex. We have now generated and characterized a Tuba8 knockout mouse model. Homozygous mice were confirmed to lack Tuba8 protein in the testis, but did not display PMG and appeared to be neurologically normal. In response to this finding, we re-analyzed the human PMG subjects using whole exome sequencing. This resulted in identification of an additional homozygous loss-of-function mutation in SNAP29, suggesting that SNAP29 deficiency, rather than TUBA8 deficiency, may underlie most or all of the neurodevelopmental anomalies in these subjects. Nonetheless, in the mouse brain, Tuba8 specifically localised to the cerebellar Purkinje cells, suggesting that the human mutations may affect or modify motor control. In the testis, Tuba8 localisation was cell-type specific. It was restricted to spermiogenesis with a strong acrosomal localization that was gradually replaced by cytoplasmic distribution and was absent from spermatozoa. Although the knockout mice were fertile, the localisation pattern indicated that Tuba8 may have a role in spermatid development during spermatogenesis, rather than as a component of the mature microtubule-rich flagellum itself.

No MeSH data available.


Related in: MedlinePlus

Generation of a Tuba8 knockout allele.A) Design of the targeting construct. A 9.1-kb SbfI–NgoAIV genomic fragment was directionally cloned into the SbfI+AgeI-restricted pUC19 vector. The shaded numbered boxes depict exons 2–4. The arrows represent the location and direction of expression of the selectable markers. The neomycin (neo) cassette, obtained from PGKneobpA [22], was inserted at the PspoMI site. The FRT sites (shaded arrow heads) flanked the neomycin positive selection cassette, whilst one LoxP site flanked one side of the neo, and the second was inserted into the BbvCI site in intron 3 (unshaded arrow heads). The diphtheria toxin negative selection cassette, PGK-DTA (kindly provided by P. Soriano, Fred Hutchinson Cancer Research Center, Seattle, WA), was inserted between the SalI and NotI sites of the modified pUC19 backbone. B) Quantitative reverse transcription PCR. Tuba8 mRNA levels in testis were determined from four animals of each Tuba8 genotype, wild type (+/+), heterozygous (+/−) and homozygous (−/−), and normalised to Hprt levels. Bars indicate mean ± 1 s.d. Statistical significance was reached for all combinations of genotypes using an unpaired t-test (p<0.05). C) Protein analysis using western blotting. Protein lysates from testis samples used four animals of each genotype: wild type (+/+), heterozygous (+/−) and homozygous (−/−), in lanes 1–3 respectively, were probed with the Bioserv Tuba8 mouse monoclonal antibody. A band of approximately 55 kDa was detected in the wild type and heterozygous samples. The blots were subsequently probed for Gapdh expression as a loading control to quantify expression.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384676&req=5

pone.0174264.g001: Generation of a Tuba8 knockout allele.A) Design of the targeting construct. A 9.1-kb SbfI–NgoAIV genomic fragment was directionally cloned into the SbfI+AgeI-restricted pUC19 vector. The shaded numbered boxes depict exons 2–4. The arrows represent the location and direction of expression of the selectable markers. The neomycin (neo) cassette, obtained from PGKneobpA [22], was inserted at the PspoMI site. The FRT sites (shaded arrow heads) flanked the neomycin positive selection cassette, whilst one LoxP site flanked one side of the neo, and the second was inserted into the BbvCI site in intron 3 (unshaded arrow heads). The diphtheria toxin negative selection cassette, PGK-DTA (kindly provided by P. Soriano, Fred Hutchinson Cancer Research Center, Seattle, WA), was inserted between the SalI and NotI sites of the modified pUC19 backbone. B) Quantitative reverse transcription PCR. Tuba8 mRNA levels in testis were determined from four animals of each Tuba8 genotype, wild type (+/+), heterozygous (+/−) and homozygous (−/−), and normalised to Hprt levels. Bars indicate mean ± 1 s.d. Statistical significance was reached for all combinations of genotypes using an unpaired t-test (p<0.05). C) Protein analysis using western blotting. Protein lysates from testis samples used four animals of each genotype: wild type (+/+), heterozygous (+/−) and homozygous (−/−), in lanes 1–3 respectively, were probed with the Bioserv Tuba8 mouse monoclonal antibody. A band of approximately 55 kDa was detected in the wild type and heterozygous samples. The blots were subsequently probed for Gapdh expression as a loading control to quantify expression.

Mentions: Tuba8-containing genomic clones were isolated from mouse strain 129S6/SvEvTac by screening the RPCI-21 PAC library (Medical Research Council (MRC) Geneservice). A fragment containing exons 2–4 was cloned into pUC19 (Fig 1a). Positive and negative selection cassettes were added, with FRT sites positioned to allow deletion of the positive selection cassette, and LoxP sites for deletion of Tuba8 exon 3 (Fig 1a).


A tubulin alpha 8 mouse knockout model indicates a likely role in spermatogenesis but not in brain development
Generation of a Tuba8 knockout allele.A) Design of the targeting construct. A 9.1-kb SbfI–NgoAIV genomic fragment was directionally cloned into the SbfI+AgeI-restricted pUC19 vector. The shaded numbered boxes depict exons 2–4. The arrows represent the location and direction of expression of the selectable markers. The neomycin (neo) cassette, obtained from PGKneobpA [22], was inserted at the PspoMI site. The FRT sites (shaded arrow heads) flanked the neomycin positive selection cassette, whilst one LoxP site flanked one side of the neo, and the second was inserted into the BbvCI site in intron 3 (unshaded arrow heads). The diphtheria toxin negative selection cassette, PGK-DTA (kindly provided by P. Soriano, Fred Hutchinson Cancer Research Center, Seattle, WA), was inserted between the SalI and NotI sites of the modified pUC19 backbone. B) Quantitative reverse transcription PCR. Tuba8 mRNA levels in testis were determined from four animals of each Tuba8 genotype, wild type (+/+), heterozygous (+/−) and homozygous (−/−), and normalised to Hprt levels. Bars indicate mean ± 1 s.d. Statistical significance was reached for all combinations of genotypes using an unpaired t-test (p<0.05). C) Protein analysis using western blotting. Protein lysates from testis samples used four animals of each genotype: wild type (+/+), heterozygous (+/−) and homozygous (−/−), in lanes 1–3 respectively, were probed with the Bioserv Tuba8 mouse monoclonal antibody. A band of approximately 55 kDa was detected in the wild type and heterozygous samples. The blots were subsequently probed for Gapdh expression as a loading control to quantify expression.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384676&req=5

pone.0174264.g001: Generation of a Tuba8 knockout allele.A) Design of the targeting construct. A 9.1-kb SbfI–NgoAIV genomic fragment was directionally cloned into the SbfI+AgeI-restricted pUC19 vector. The shaded numbered boxes depict exons 2–4. The arrows represent the location and direction of expression of the selectable markers. The neomycin (neo) cassette, obtained from PGKneobpA [22], was inserted at the PspoMI site. The FRT sites (shaded arrow heads) flanked the neomycin positive selection cassette, whilst one LoxP site flanked one side of the neo, and the second was inserted into the BbvCI site in intron 3 (unshaded arrow heads). The diphtheria toxin negative selection cassette, PGK-DTA (kindly provided by P. Soriano, Fred Hutchinson Cancer Research Center, Seattle, WA), was inserted between the SalI and NotI sites of the modified pUC19 backbone. B) Quantitative reverse transcription PCR. Tuba8 mRNA levels in testis were determined from four animals of each Tuba8 genotype, wild type (+/+), heterozygous (+/−) and homozygous (−/−), and normalised to Hprt levels. Bars indicate mean ± 1 s.d. Statistical significance was reached for all combinations of genotypes using an unpaired t-test (p<0.05). C) Protein analysis using western blotting. Protein lysates from testis samples used four animals of each genotype: wild type (+/+), heterozygous (+/−) and homozygous (−/−), in lanes 1–3 respectively, were probed with the Bioserv Tuba8 mouse monoclonal antibody. A band of approximately 55 kDa was detected in the wild type and heterozygous samples. The blots were subsequently probed for Gapdh expression as a loading control to quantify expression.
Mentions: Tuba8-containing genomic clones were isolated from mouse strain 129S6/SvEvTac by screening the RPCI-21 PAC library (Medical Research Council (MRC) Geneservice). A fragment containing exons 2–4 was cloned into pUC19 (Fig 1a). Positive and negative selection cassettes were added, with FRT sites positioned to allow deletion of the positive selection cassette, and LoxP sites for deletion of Tuba8 exon 3 (Fig 1a).

View Article: PubMed Central - PubMed

ABSTRACT

Tubulin alpha 8 (Tuba8) is the most divergent member of the highly conserved alpha tubulin family, and uniquely lacks two key post-translational modification sites. It is abundantly expressed in testis and muscle, with lower levels in the brain. We previously identified homozygous hypomorphic TUBA8 mutations in human subjects with a polymicrogyria (PMG) syndrome, suggesting its involvement in development of the cerebral cortex. We have now generated and characterized a Tuba8 knockout mouse model. Homozygous mice were confirmed to lack Tuba8 protein in the testis, but did not display PMG and appeared to be neurologically normal. In response to this finding, we re-analyzed the human PMG subjects using whole exome sequencing. This resulted in identification of an additional homozygous loss-of-function mutation in SNAP29, suggesting that SNAP29 deficiency, rather than TUBA8 deficiency, may underlie most or all of the neurodevelopmental anomalies in these subjects. Nonetheless, in the mouse brain, Tuba8 specifically localised to the cerebellar Purkinje cells, suggesting that the human mutations may affect or modify motor control. In the testis, Tuba8 localisation was cell-type specific. It was restricted to spermiogenesis with a strong acrosomal localization that was gradually replaced by cytoplasmic distribution and was absent from spermatozoa. Although the knockout mice were fertile, the localisation pattern indicated that Tuba8 may have a role in spermatid development during spermatogenesis, rather than as a component of the mature microtubule-rich flagellum itself.

No MeSH data available.


Related in: MedlinePlus