|
Gene-insertion cassette
|
ssDNA length Left Arm-Cassette-Right Arm (bases) [source of ssDNA]
|
Zygotes injected
|
Zygotes transferred
|
Live-born animals (percentage of transferred zygotes)
|
Targeted animals (%)a
|
|---|
|
Pitx1-exon 2 floxed
|
93 + 862 + 91
[IvTRT]
|
85
|
76
|
10 (13.2)
|
4 (40%)b
|
|
Ambra1-exon 4 floxed
|
96 + 589 + 103
[IvTRT]
|
67
|
63
|
8 (12.7)
|
6 (75%)c
|
|
Col12a1-exon 2 floxed
|
55 + 527 + 55
[IvTRT]
|
105
|
79
|
3 (3.8)
|
3 (100%)d
|
|
Ubr5-exon 58 floxed
|
78 + 535 + 86
[IvTRT]
|
20
|
16
|
2 (12.1)
|
2 (100%)e
|
|
Syt1-exon 6 floxed
|
75 + 635 + 75
[IDT Megamer™]
|
51
|
45
|
8 (17.8)
|
1 (12.5%)f
|
|
Syt9-exon 3 floxed
|
87 + 893 + 68
[IDT Megamer™]
|
43
|
41
|
12 (29.3)
|
1 (8.5%)g
|
|
PPP2r2a-exon 3 floxed
|
95 + 619 + 84
[IDT Megamer™]
|
34
|
33
|
3 (9.1)
|
3 (100%)h
|
-
aThe alleles that did not contain the inserts were not analyzed for the presence of indels because genotyping assays were mainly designed to identify the targeted-insertion alleles. However, noticeable deletions were observed for some samples (e.g., deletions in the non-targeted alleles; Fig. 2g, h; Additional file 1: Figure S2i)
-
bAnimals 3, 5, 7, and 8 were heterozygous for both 5′ and 3′ LoxP sites. Animal 5 had a floxed allele with one nucleotide insertion mutation at the intronic region, which may not affect function. Animals 2, 9, and 10 had only 5′ LoxP site, and animal 4 had only 3′ LoxP site (Fig. 1g)
-
cAnimals 1, 2, 3, 5, 7, and 8 were heterozygous for both the 5′ and 3′ LoxP sites (Fig. 2g). Animal 7 had a floxed allele with 1-bp insertion mutation in the intronic region, which may not affect function
-
dAnimals 1 and 2 were heterozygous for both 5′ and 3′ LoxP sites and they carried deletions in their second allele. Animal 3 was biallelic for both LoxP sites (Fig. 2h)
-
eAnimals 1 and 2 were heterozygous for both 5′ and 3′ LoxP sites (Fig. 2i)
-
fAnimals 4 and 7 had only 5′ LoxP insertion and the animal 6 had correctly targeted LoxP sites (Additional file 1: Figure S2g)
-
gAnimal 12 had correctly targeted LoxP sites and all others were wild type (Additional file 1: Figure S2h).
-
hAnimals 1 and 2 were heterozygous for both loxPs with deletions in the second allele and pup 3 was biallelic (Additional file 1: Figure S2i)
