Background We have developed and validated a set of complementary retroviruses that creates a wide range of nested chromosomal deletions. When applied to mouse ES cells, this retrovirus-based method generated deletions ranging from 6 kb to 23 Mb (average 2.9 Mb), with an efficiency of 64% for drugs-selected clones. Importantly, several of engineered ES cell clones, mostly those with large deletions, showed major alteration in cell fate.
The set of complementary replication-defective retroviruses exploited the Cre-loxP recombination system and reconstitution of a functional neomycin cassette for selection of recombination events. The first loxP sequenced was delivered using the anchor virus A1. The integration was selected on puromycin. ES cell clones containing one copy of the virus A1 were isolated and expanded (primary clones). A second loxP was introduced by retroviral gene transfer using the saturation virus S1, selected on hygromycin (secondary clones). The transient expression of the recombinase Cre in secondary clones allowed the recombination between the loxP sites and the functional reconstitution of a split neomycin expression cassette. Neomycin resistant clones (tertiary clones) were isolated and expanded. Chromosomal deletions are expected to have occurred in neomycin resistant clones that have lost both puromycin and hygromycin resistance genes. In addition, other chromosomal rearrangements (e.g., inversions, translocations, etc.) are achievable using this system.
Aim Inverse-PCR, array-based comparative genomic hybridization (aCGH) and spectral karyotyping were employed to confirm deletions (or other Cre-induced rearrangements) in several tertiary clones and to assess the genomic integrity of the ES cells altered.
Conclusions aCGH conclusions are summarized together with complementary results from inverse-PCR and spectral karyotyping in 3 tables: Table 1: Summary of the virus A1 integration Table 2: Characteristics of independent deletions confirmed by IPCR-aCGH Table 3: Confirmed or suspected recombination events in trans
Notes for Table 2: Mapping and deletion analyses were done using the UCSC Genome Browser (NCBI mouse Build 33). {a} Tertiary clones are labeled according to their family number (same integration of virus A1), followed by a specific id number. If more than one clone presented a redundant rearrangement within the same group infected with virus S1, only one is reported for clarity. {b} Anomaly that was not present in the primary clone from which the tertiary clone was derived, as determined by aCGH or SKY. ( -), no anomaly; (+), additional anomaly. {c} The deletion is not observed, in agreement with the resolution of aCGH. {d} Normal except for the loss of chromosome Y. {e} Amplification of chromosome 1. {f} Amplification of chromosome 8. {g} Many chromosomes were lost according to SKY. {h} Amplification on chromosome 14. Id, identification; kb, kilobase pairs; no., number; aCGH, array-based comparative genomic hybridization; SKY, spectral karyotyping; I-PCR, inverse-PCR; n.d., not determined.
Keywords: comparative genomic hybridization
Overall design
aCGH (n=30) were conducted between our mouse ES cell samples and a control normal mouse genomic DNA. Selected tertiary clones (n= 20) were compared to their corresponding primary clones (n=9), in order to find anomalies that was not present in the primary clones from which they were derived. Primary clones were compared together and to the unmodified R1 ES cells (n=1) to assess the genomic integrity.
Table 1: Summary of the virus A1 integration ({a} Also tested by SKY: 8 mitoses 40,XY and 4 mitoses 39,X,-Y out of 15 analyzed. {b} Trisomy chromosome 1. {c} Amplification and deletion on chromosome 4, position 52-76 Mb and an amplification of ~7Mb at the telomeric end of chromosome X. {d} Normal except the loss of a BAC on chromosome 2, position 164 Mb. Id, identification; N, normal; A, abnormal; n.d., not determined) header descriptions
Primary clone id
Chromosome (I-PCR)
Start coordinate (I-PCR)
End coordinate (I-PCR)
aCGH
Data table
Primary clone id
Chromosome (I-PCR)
Start coordinate (I-PCR)
End coordinate (I-PCR)
aCGH
1
14
22164853
22165099
N
2
5
63019373
63019748
N
4
2
167486315
167486681
N
6
17
27622141
27622184
N
7
16
36011960
36012543
N
9
18
57155297
57155985
N {a}
10
16
65165857
65166035
A {b}
12
X
40497593
40497726
n.d.
13
4
83558072
83558421
A {c}
14
2
156503302
156503387
N {d}
15
11
68627686
68627918
n.d.
Total number of rows: 11
Table 3: Confirmed or suspected recombination events in trans ({a} Clones showing rearrangement redundancy are on the same lane. {b} aCGH only for 14-39. ID, identification; Puro, puromycin; Hygro, hygromycin; S,sensitive; R, resistant; n.d., not determined) header descriptions
Tertiary clone ID {a}
Puro
Hygro
Virus A1 integration Chromo-some
Virus A1 integration Start coordinate
Virus A1 integration End coordinate
Virus S1 integration Chromo-some
Virus S1 integration Start coordinate
Virus S1 integration End coordinate
Karyotype performed by
Event in trans Confirmed or Suspected
Data table
Tertiary clone ID {a}
Puro
Hygro
Virus A1 integration Chromo-some
Virus A1 integration Start coordinate
Virus A1 integration End coordinate
Virus S1 integration Chromo-some
Virus S1 integration Start coordinate
Virus S1 integration End coordinate
Karyotype performed by
Event in trans Confirmed or Suspected
2-03
S
R
5
63019373
63019748
19
6920757
6921025
n.d.
C
4-03
S
R
2
167486315
167486681
n.d.
n.d.
n.d.
SKY
S
4-09
S
R
2
167486315
167486681
n.d.
n.d.
n.d.
n.d.
S
9-36
R
S
18
57155297
57155985
n.d.
n.d.
n.d.
n.d.
S
9-40
R
S
18
57155297
57155985
n.d.
n.d.
n.d.
n.d.
S
9-62
S
S
18
57155297
57155985
18
77983260
77983459
SKY and aCGH
S
14-01 and 14-39
S
R
2
156503302
156503387
n.d.
n.d.
n.d.
aCGH {b}
S
14-27
S
S
2
156503302
156503387
16
16998336
16998873
n.d.
C
14-32
S
R
2
156503302
156503387
16
16998336
16998899
SKY and aCGH
C
Total number of rows: 9
Table 2: Characteristics of independent deletions confirmed by IPCR-aCGH (for legends, see the summary section above) header descriptions
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