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Series GSE6706 Query DataSets for GSE6706
Status Public on Jan 13, 2007
Title A retroviral strategy that efficiently creates chromosomal deletions in mammalian cells
Organism Mus musculus
Experiment type Genome variation profiling by genome tiling array
Summary 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.
 
Contributor(s) Bilodeau M, Girard S, Hébert J, Sauvageau G
Citation(s) 17277782
Submission date Jan 10, 2007
Last update date Mar 16, 2012
Contact name Guy Sauvageau
E-mail(s) guy.sauvageau@gmail.com
Organization name IRIC
Department Université de Montréal
Lab Laboratory of Molecular Genetics of Stem Cells
Street address Marcelle-Coutu Building 2900 Boul. Edouard Montpetit Quai 20
City Montreal
State/province Quebec
ZIP/Postal code H3T 1J4
Country Canada
 
Platforms (1)
GPL4714 RPCI Mouse 6.5K
Samples (30)
GSM154569 ES cells primary clone no 1
GSM154570 ES cells tertiary clone no 1-03
GSM154571 ES cells tertiary clone no 1-13
Relations
BioProject PRJNA99023

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
Tertiary clone id {a}
Data source
Chromo-some
Start coordinate
End coordinate
Size of deletions (kb)
aCGH confirmation
Genomic anomaly {b}

Data table
Tertiary clone id {a} Data source Chromo-some Start coordinate End coordinate Size of deletions (kb) aCGH confirmation Genomic anomaly {b}
1-3 I-PCR 14 22165099 23710534 1545 (+) (-)
1-13 I-PCR 14 22165099 44937742 22773 (+) (-)
4-2 I-PCR 2 167486681 168900222 1414 n.d. n.d.
6-36 I-PCR 17 26955839 27622141 666 (+) (+) {d,e}
7-30 I-PCR 16 35918443 36011960 94 (+) {c} (-)
9-31 I-PCR 18 57155985 57162362 6 n.d. n.d.
9-107 I-PCR 18 57155985 57166502 10 n.d. n.d.
9-71 I-PCR 18 57155985 57174937 19 n.d. n.d.
9-17 I-PCR 18 57155985 57174941 19 n.d. (-) {d}
9-68 I-PCR 18 57155985 57175174 19 n.d. n.d.
9-29 I-PCR 18 57155985 57177486 22 n.d. n.d.
9-35 I-PCR 18 57155985 57179077 23 (+) {c} (-)
9-90 I-PCR 18 57155985 57473132 317 (+) {c} (+) {f}
9-104 I-PCR 18 57155985 61338307 4182 n.d. (+) {g}
9-37 I-PCR 18 57155985 61468765 4313 (+) (-) {d}
9-18 I-PCR 18 57155985 62204954 5049 (+) (-)
10-18 I-PCR 16 59749084 65165857 5417 (+) (-)
10-21 I-PCR 16 57307345 65165857 7858 (+) (+) {h}
13-34 aCGH 4 78266064 82222600 3956 (+) (-)
14-16 I-PCR 2 156503387 157071542 568 (+) {c} (-)

Total number of rows: 22

Table truncated, full table size <1 Kbytes.




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