1
Supporting Methods
2
3
Creating of the targeting construct to generate the Acta1(H40Y) KI mouse line
4
The 5.1kb and the 2.3kb fragments were PCR amplified from total genomic DNA obtained
5
from wt/wt ES (129/Sv) using the following primers: 5’AGGTGTGCGGCCGCAGGTTT
6
CCTGTAG3’ and 5’ACGGCCAGCGGCCGCCTGTTCGCA3’ for the 5.1kb fragment and
7
5’CTTCCTATCGATCAAATGCTGGTGGCAC3’
8
CCCTA3’ for the 2.3kb fragment. This amplification introduces a NotI and a ClaI restriction
9
site into the 5.1kb and the 2.3kb fragments, respectively. The NotI digested 5.1kb fragment
10
and ClaI digested 2.3kb fragment were cloned into linearized pBluescript and pPGK-Neo-
11
DTA vectors, respectively, to generate pBluescript-5.1kb and pPGK-Neo-DTA-2.3kb
12
plasmids. Correct orientation of the inserted fragments was confirmed and the 5.1kb and
13
2.3kb inserts were sequenced to ensure that no mutations or rearrangements were introduced
14
by PCR and the cloning procedures.
and
5’AGCTTCATGGTGCCCTCT
15
16
A 3 step PCR-based approach was used to generate the site-directed mutagenesis of the Acta1
17
sequence in the pBluescript-5.1kb plasmid. Primers (5’CGTGGGCCGACCCCGTT
18
ACCAGGTCAGGCTGCTGGCAGGG3’ and 5’CCCTGCCAGCAGCCTGACCTGGTAAC
19
GGGGTCGGCCCACG3’) were designed to bind to either sides of the mutation site in
20
pBluescript-5.1kb to introduce the single nucleotide base change A to G. This single-base
21
-skeletal actin
22
protein.
The
23
5’TCCCCTGCATGCCTAGTTGGA3’ and 5’CCCTGCCAGCAGCCTGACCTGGTAACG
24
GGGTCGGCCCACG3’; 5’CTTCTGCATGCGGTCAGCGATAC3’ and 5’CGTGGGCCG
25
ACCCCGTTACCAGGTCAGGCTGCTGGCAGGG3’) on the pBluescript-5.1kb template to
26
generate 400bp and 1.3kb fragments, respectively. The third PCR reaction was performed on
27
DNA
from
first
the
two
first
PCR
two
reactions
reactions
1
were
using
performed
the
(primer
following
pairs
primers:
28
5’TCCCCTGCATGCCTAGTTGGA3’
and
5’CTTCTGCATGCGGTCAGCG
ATAC3’.
29
These two primers were designed to bind on either side of the mutation, across a SphI
30
restriction site to allow for the cloning of the PCR mutated fragment back into the
31
pBluescript-5.1kb plasmid. The resultant 1.7kb PCR fragment was extracted from a 1.2%
32
agarose gel, linearized with SphI and then ligated into a SphI digested pBluescript-5.1kb. The
33
entire mutated sequence within pBluescript-5.1kb was sequenced to ensure there were no
34
additional PCR-induced mutations or cloning-induced rearrangements.
35
36
The mutated 5.1kb fragment was removed from pBluescript-5.1kb using NotI and was then
37
inserted into NotI linearized pPGK-Neo-DTA-2.3kb to create the complete KI construct. PCR
38
(primers 5’GCTTGCCTGGTCCCTGGCTGCTTG3’ and 5’GTGGTTGTGTGTTGTCCTA
39
G3’) was used to confirm the correct orientation of the 5.1kb fragment.
40
41
In-Gel digestion and matrix-assisted laser desorption ionization time-of-flight mass
42
spectrometry (MALDI-TOF MS) analysis
43
The MALDI-
44
skeletal muscles of ACTA1(H40Y) KI mice. Total protein was extracted from the skeletal
45
muscles of an 8 week-old KI mouse as described previously.24 Two microlitres of total
46
protein were fractionated through a 12.5% SDS-PAGE gel and protein bands were visualised
47
with Colloidal Coomassie staining (BioRad, Regents Park, Australia). The α-skeletal actin
48
protein band was excised from the gel and processed as previously described.25, 26 A Voyager-
49
DE PRO matrix assisted laser desorption ionization time-of-flight mass spectrometer (Applied
50
Biosystems, Scoresby Vic, Australia) equipped with delayed extraction was employed for
51
peptide mapping in positive reflector mode. Database search and protein identification was
52
done with In-house Mascot search engine (www.matrixscience.com), and all post-acquisition
-skeletal actin in
2
53
data analysis and calculations were performed using Data Explorer TM software (Applied
54
Biosystems).
55
56
57
58
59
60
61
62
63
64
65
66
67
Supplemental Figure 1. Acta1(H40Y) KI construct and screening strategies. (A) The KI
targeting construct is shown in black. The PGK-Neo sequence is flanked on either side by
Cre-lox P recombination sites (triangles). Downstream of the PGK-Neo gene is an additional
2.3kb genomic DNA sequence that is at the 3’ end of the Acta1 gene. Upstream of the PGKNeo sequence is 5.1kb of DNA sequence from Acta1, including 1.8kb sequence upstream of
exon 1. The PCR-generated mutation occurs in exon 2 of Acta1. The placement of the 3
probes designed for Southern screening and the locations of EcoRI restriction sites are shown.
(B) DNA samples extracted from embryonic stem cells (ESC) were digested with EcoRI
before Southern analyses. An example of a Southern blot of ESC is shown. Clone #65 (*)
contains the smaller knock-in (ki) allele when screened with the 3 probes. The WT (wt) allele
gives a larger 7.3kb band. All KI mice described in this study were derived from this clone.
3
68
69
70
71
72
73
74
75
Genomic DNA was extracted from the gastrocnemius muscles of the 9 chimaeric mice that
died and screened for the neomycin sequence (C) and sequenced to determine the presence of
the single base pair mutation (*) (D). (E) MALDI-TOF MS detection of mutant (H40Y) and
WT α-skeletal actin peptides in skeletal muscle from the KI mice.
Supplemental Figure 2. (A) Absolute twitch and (B) tetanic (200Hz) force output in isolated
EDL muscles from WT (wt/wt) and KI (ki/wt) 8 week old mice (n = 5-7 mice/group). (C)
Specific twitch and (D) tetanic (150Hz) force output in isolated soleus muscles from 8 week
4
76
77
78
79
80
81
82
83
84
85
86
old WT (wt/wt) and KI (ki/wt) mice (n = 5-6 mice/group). (E) Body weights of 8 week old
female WT, KI and KI mice crossed with the c-ski (ki/c-ski), FHL1 (ki/FHL1) and IGF-I
(ki/IGF-I) transgenic mice (n = 7-23 mice/group). (F) Gastrocnemius and (G) EDL muscle
weights in 8 week old female WT (wt/wt), KI (ki/wt) and KI mice crossed with the c-ski,
FHL1 and IGF-I transgenic mice (n = 4-11 mice/group). (H) Forearm grip strength of female
WT, KI and KI mice crossed with the c-ski transgenic mice (n = 4-28 mice/group). (I) The
number of ambulations over a 20 minute period in an Open Filed apparatus for female WT,
KI and KI mice crossed with the c-ski transgenic mice (n = 6-22 mice/group). For all graphs
(A-I), data are expressed as mean ± SE. Statistical significance from the wt/wt mice are
indicated by + (P < 0.05) and significance compared to the ki/wt mice are indicated by * (P <
0.05).
5