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First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00768

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A targeted deletion of the C-terminal end of titin, including the titin kinase domain, impairs myofibrillogenesis

Gaynor Miller^1,*, Hanny Musa¹, Matthias Gautel² and Michelle Peckham^1,

¹ School of Biomedical Sciences, University of Leeds, LS2 9JT, UK
² Muscle Cell Biology, The Randall Centre, King's College London, London, SE1 1UL, UK

View larger version (26K): [in a new window]   Fig. 1. This diagram shows the strategy used to target the titin gene. An XhoI-SalI fragment containing the Pgk-Neo gene was cloned into the SalI site (S), as shown (black box shows position of pgk promoter). The ThK gene was cloned outside the genomic DNA as shown. (A) Diagram of the titin 10 kb genomic DNA cloned into PCRIIM. Shaded regions are the approximate positions of exons; unshaded regions are the approximate positions of introns; lines indicate bacterial vector backbone; stippled regions represent regions of the titin gene that are outside the region of genomic DNA used to generate the targeting vector; filled boxes represent the position of the promoters for the Pgk-Neo and ThK genes; the box with square hatching represents the Neo gene; the box with the dotted diamond pattern represents the ThK gene. Letters represent restriction sites as follows: E, EcoRI; H, HindIII; S, SalI; N, NotI; X, XhoI. (B) The targeting DNA was digested using a unique NotI site to produce the linearised targeting DNA as shown. This linearised DNA was electroporated into cells, where it recombined with the endogenous DNA at the genomic locus as shown, replacing the endogenous titin allele. The positions of the pair of PCR primers used for initial screening to identify targeted cells is shown, together with the position of a 1.5 kb BamHI-SalI DNA fragment used in subsequent southern analysis of the genomic DNA.

View larger version (90K): [in a new window]   Fig. 2. PCR analysis and Southern blot. (A) PCR analysis of genomic DNA used to initially screen for targeted cells. The gel shows duplicate reactions for 4 separate clones (16, 20, 25 and 68-G) that were the only clones to give positive bands by this PCR screen. Clone 16 (lanes 1and 2), clone 20 (lanes 3 and 4), clone 25 (lanes 5 and 6), clone 68-G (lanes 7 and 8) and lane 9 is a negative control. (B) Subsequent southern blot for 3 of clone 20 (lane 1), clone 25 (lane 2), clone 68-G (lane 3) and wild-type (lane 4) cells. The blot was probed using a genomic DNA probe that was outside the targeted region (see Fig. 1). Clone 20 is the only clone that shows a targeted band as well as a wild-type band, with no other additional bands. We expected that this probe would detect a band of 5.7 kb in an untargeted allele, and that the size of this band would increase to 7.2 kb, if the allele had been targeted.

View larger version (30K): [in a new window]   Fig. 3. SDS gel electrophoresis of myotube proteins. (A) A 3-7.5% gradient protein gel for the targeted clone (lanes 1-3, separate protein samples) and wild-type clone (lane 4) that has been silver stained. For both wild-type and the targeted (c20) clones, two bands for titin can be seen. In the targeted clone, the lower band is approximately 0.2 MDa smaller than the corresponding band in the wild-type clone. This reduction in size is consistent with the targeted deletion of the C-terminal end. Protein samples were not equally loaded for this gel resulting in some intensity variation for the bands for c20 samples. Approximate molecular masses are shown on the right, in MDa. (B) A western blot of 2-7.5% gradient protein gel for heart muscle (lane 1), wild-type 5-day myotubes (lane 2), and targeted (c20) 5-day myotubes (lane 3) using the Z1/Z2 antibody, against the N-terminal domain of titin. The isoform expressed in heart muscle is approximately 2.8-2.9 MDa, and can be seen as a single band. In the myotubes, there are two immunopositive bands for titin in both the wild-type and the targeted (c20) clones. The difference in sizes for the two bands in the wild-type and targeted clones cannot be resolved on this blot. (C) A western blot of a 2-7.5% gradient protein gel for wild-type (lanes 1 and 3; two separate protein samples), and clone 20 (lanes 2 and 4; two separate protein samples). Two bands for titin kinase can be seen in both the wild-type samples, however, only one band can be seen in both c20 samples when probed with the anti-kinase antibody. This gel was run for longer than the gel in B, hence the larger separation between the two bands present in the wild-type clone. This suggests that the lower band in c20 is not immunoreactive to the kinase antibody.

View larger version (117K): [in a new window]   Fig. 5. Two representative fields at low magnification, of (A) wild-type and (B) targeted 5-day myotubes cultures, immunostained for skeletal myosin. Scale bar: 20 µm.

View larger version (34K): [in a new window]   Fig. 4. Bar charts showing lengths of myotubes in wild-type and targeted (c20) clones (n=50 for each). Lengths were grouped as shown on the x axis. This chart shows that the myotubes in the targeted clone (c20) tend to be shorter than those in the wild-type clone.

View larger version (86K): [in a new window]   Fig. 6. Confocal images at high magnification, of wild-type (A,C,E,G) and targeted (c20) cells, immunostained for titin, myosin and -actinin. Myotubes were immuno-stained for skeletal myosin (A,B), anti-skeletal -actinin (C,D), anti-Z-line titin (E,F) and anti-M-line titin (G,H). Scale bar: 20 µm.

View larger version (64K): [in a new window]   Fig. 7. Confocal images at high magnification, of wild-type and targeted (c20) cells co-immunostained for myomesin and telethonin (A-F); skeletal myosin and MURF2 (G-L), or skeletal myosin and obscuring (M-R). The staining for each antibody is shown separately, together with the merged image. Arrow in H indicates microtubular-like staining; arrows in O indicate M-line staining. Both wild-type and c20 cultures contain myotubes at a variety of stages of differentiation as seen here, with the myotubes in the central region typical of the most highly differentiated. Scale bar: 10 µm.