Open Access

Assessment of genome integrity with array CGH in cattle transgenic cell lines produced by homologous recombination and somatic cell cloning

Genome Integrity20112:6

https://doi.org/10.1186/2041-9414-2-6

Received: 18 January 2011

Accepted: 23 May 2011

Published: 23 May 2011

Abstract

Background

Transgenic cattle carrying multiple genomic modifications have been produced by serial rounds of somatic cell chromatin transfer (cloning) of sequentially genetically targeted somatic cells. However, cloning efficiency tends to decline with the increase of rounds of cloning. It is possible that multiple rounds of cloning compromise the genome integrity or/and introduce epigenetic errors in the resulting cell lines, rendering a decline in cloning. To test these possibilities, we performed 9 high density array Comparative Genomic Hybridization (CGH) experiments to test the genome integrity in 3 independent bovine transgenic cell lineages generated from genetic modification and cloning. Our plan included the control hybridizations (self to self) of the 3 founder cell lines and 6 comparative hybridizations between these founders and their derived cell lines with either high or low cloning efficiencies.

Results

We detected similar amounts of differences between the control hybridizations (8, 13 and 39 differences) and the comparative analyses of both "high" and "low" cell lines (ranging from 7 to 57 with a mean of ~20). Almost 75% of the large differences (>10 kb) and about 45% of all differences shared the same type (loss or gain) and were located in nearby genomic regions across hybridizations. Therefore, it is likely that they were not true differences but caused by systematic factors associated with local genomic features (e.g. GC contents).

Conclusions

Our findings reveal that large copy number variations are less likely to arise during genetic targeting and serial rounds of cloning, fortifying the notion that epigenetic errors introduced from serial cloning may be responsible for the cloning efficiency decline.

Keywords

genome integrity cattle transgenic cell line somatic cell cloning array CGH

Findings

As embryonic stem cells are not available in the bovine species, somatic cells have been used for genetic modifications, and transgenic cattle have been produced from such genetically modified somatic cells by animal cloning. However, because primary somatic cells have limited life span and inevitably become senescent following DNA transfection and selection in cell culture, it is impossible to perform any further genetic modifications in these cells. Because of such, transgenic cattle with a desired genotype that requires more than one genetic targeting event, such as homozygous deletion of the two alleles of a gene, cannot be produced. To overcome such limitations, a novel sequential genetic modification strategy in bovine somatic cells, for producing extensively genetically modified cattle, has been developed [1]. This process involves a serial round of genetic targeting events, each followed by cloning to rejuvenate the genetically modified somatic cells (to rescue them from senescence) for the next round of genetic targeting. Such genetically modified somatic cells are then subjected to a final round of cloning for producing transgenic animals with the desired genotypes. While multiple genomic loci have been modified by this strategy, cloning efficiency tends to decline with the increased rounds of cloning, and in some severe cases, such manipulated cells can become unclonable (no live calf can be cloned from them) [2]. It is yet unknown whether the cloning efficiency declines in such derived cells are due to genetic abnormalities caused by the multiple genetic targeting or/and serial cloning process or due to the accumulation of epigenetic errors introduced during these processes. Such questions are fundamental in farm animal transgenesis, as somatic cells and cloning are currently the only choices for genetic modifications and for transgenic animal production in the domestic animal species.

To investigate whether the declines of cloning efficiency in the cloned bovine transgenic cell lines are due to large genomic deletions or insertions, 9 high density array Comparative Genomic Hybridization (CGH) experiments were performed to test the genome integrity in 3 independent bovine transgenic cell lineages. Array CGH allows the entire genome to be assayed for the gain or loss of material in a single experiment by measuring the relative hybridization intensity between fluorescently labeled test and reference DNA samples. It has been widely used in the detection of copy number variations (CNVs). One objective of this study is to develop array CGH into a systematic test for the genomic integrity of donor cells after each round of genetic modification before they are used as donors for producing transgenic animals.

We selected 3 independent cell lineages from our transgenic bovine cell line collection. Each lineage includes the founder and two derived cell lines, which demonstrated dramatic differences in cloning efficiency (Figure 1). The cloning efficiencies are represented by the live calf counts at birth divided by recipient numbers used for embryo transfer as shown in parentheses. Test lines were classified into "high (H)" and "low (L)" based on their cloning efficiencies, with 7%-42% live calving rates designated as high and 0% as low. The procedures for genetic modifications, animal cloning and transgenic cell line establishment were described previously [1]. Genomic DNA samples were purified from the cell lines using Qiagen Miniprep Kit as recommended by the manufacturer. All DNA samples were analyzed by Nanodrop spectrophotometer and agarose gel electrophoresis. Nine array CGH experiments were carried out using each cell line as the test sample and the corresponding founder line as the reference sample (Table 1). Therefore, our plan included the control hybridizations of the 3 founder cell lines (self to self) and 6 comparative hybridizations between these founders and their derived cell lines of extreme phenotypes ("high" versus "low" cloning efficiencies). Another self to self control hybridization was performed using the sequenced Hereford cow L1 Dominette 01449 (Dt, American Hereford Association registration number 42190680). Each CGH array contains ~2.1 million oligonucleotide probes that provide a genome-wide coverage with an average interval of ~1.2 kb (kilo basepairs) on the UMD3 genome assemblies [3]. DNA labeling, hybridizations, array scanning, data normalization, and segmentation were performed as described before [4, 5]. The genomic variations were represented by gains and losses of normalized fluorescence intensities relative to the reference. The calls are filtered according to the similar criteria as described previously [6]. Briefly, we tested a series of log2 ratio shift and affected neighboring probe counts and their impact on the false discovery rate in the self-self control hybridizations. We then selected a calling criterion, requiring that alternations of 0.5 log2 ratios over 5 neighboring probes, under which minimal false positives were found for self-self control hybridizations. Thus, the arrays have a resolution of approximately 4.8 kb. Nine array CGH data have been submitted to the gene expression omnibus (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE26132.
Figure 1

Three cell lineages (founders and test cell lines) and their success rates for animal cloning. Live calving rates for the cell lines were calculated by the live calf counts at birth divided by recipient numbers used for embryo transfer as shown in parentheses. Cell lines with 7% or more living rates are indicated as High (H; high calving rate) and those with 0% live calving rate as Low (L; low calving rate). The 3 founder cell lines (F1, F2 and F3) were established from 3 different fetuses (day 40) respectively that were produced by artificial insemination. The 6 test cell lines, except for cell line L3, were derived from 2 rounds genetic modification and somatic cell cloning. L3 line was derived from 3 rounds of genetic modification and somatic cell cloning.

Table 1

Hybridization plan and event counts

No

Test

Ref

Type

Events

1

F1

F1

Self1

13

2

H1

F1

High1

13

3

L1

F1

Low1

7

4

F2

F2

Self2

8

5

H2

F2

High2

7

6

L2

F2

Low2

57

7

F3

F3

Self3

39

8

H3

F3

High3

17

9

L3

F3

Low3

22

10

Dt

Dt

Self4

3

We detected 8, 13 and 39 differences in 3 control hybridizations. Similar amounts of differences (ranging from 7 to 57 with a mean of ~20) were detected in comparative analyses of both "high" and "low" derived cell lines (Table 1 and Table 2). We also made event calls on Btau_4.0 and obtained a comparable number of events (data not shown). Almost 75% of the large differences (>10 kb, 42/58 events in Table 2) and about 45% of all differences (82/186 events) shared the same type (loss or gain) and were located in nearby genomic regions across hybridizations. Therefore, it is likely that they were not true differences but instead caused by systematic factors like dye bias (Cy3 versus Cy5) or genomic waves associated with local genomic features, such as GC contents [7]. For example, a variable region of chr25:27220643-27226199 from UMD3 (5.5 kb) was found in hybridizations of High1, Self3 and High3. Using liftOver, we migrated this region to its corresponding region at chr25:28829889-28835660 on Btau_4.0. The GC% track and array CGH probe track are shown in the UCSC genome browser snapshot (Figure 2). Although each probe has a GC% range from 42-48%, the average GC% of this region (53.5%) is significantly higher than the cattle genome average of 41.7% and multiple GC% peaks exist in the close proximity of 3 out of the 6 probes. Out of 186 events, 129 events are unique after merging the overlapped events (data not shown). Out of these 129 unique events, 71 events can be successfully migrated from UMD3 to Btau_4.0 and all of them showed various degrees of higher GC contents as compared to the genome average.
Table 2

Copy number variation events larger than 10 kb

No

Type

Chr

Start

End

Length

Log R

Shared

1

Self1

chr13

48,998,999

49,016,999

18,000

0.5168

Yes

  

chr3

1,020,294

1,039,699

19,405

0.6721

Yes

  

chr4

41,465,452

41,496,569

31,117

0.6946

Yes

2

High1

chr13

48,992,999

49,010,999

18,000

0.6269

Yes

  

chr3

1,020,294

1,039,699

19,405

0.6410

Yes

  

chr4

33,570,495

33,584,300

13,805

0.5216

Yes

  

chr4

41,465,452

41,496,569

31,117

0.5674

Yes

3

Low1

chr13

48,991,360

49,017,997

26,637

0.6818

Yes

  

chr29

19,400,430

19,449,274

48,844

0.5343

Yes

  

chr3

1,020,294

1,042,839

22,545

0.7566

Yes

  

chr4

41,465,452

41,496,569

31,117

0.8130

Yes

4

Self2

chr13

48,991,360

49,017,997

26,637

0.5172

Yes

  

chr3

1,020,294

1,042,839

22,545

0.6039

Yes

  

chr4

41,465,452

41,496,569

31,117

0.5761

Yes

5

High2

chr25

32,373,045

32,464,814

91,769

0.6769

Yes

6

Low2

chr11

87,532,580

87,543,090

10,510

-0.5617

No

  

chr2

16,958,057

16,968,620

10,563

-0.5814

No

  

chr25

32,374,157

32,471,634

97,477

-0.8136

Yes

  

chr29

43,204,051

43,223,301

19,250

-0.5334

No

  

chrX

10,447,331

10,457,486

10,155

-0.9137

No

7

Self3

chr1

5,249,999

5,285,999

36,000

0.5481

Yes

  

chr10

59,478,526

59,531,204

52,678

0.5501

No

  

chr13

48,991,360

49,017,997

26,637

0.6809

Yes

  

chr15

26,576,999

26,602,199

25,200

0.6474

Yes

  

chr2

39,223,655

39,235,168

11,513

-0.5661

No

  

chr25

32,374,157

32,471,634

97,477

-0.6113

Yes

  

chr29

19,399,250

19,449,274

50,024

0.6094

Yes

  

chr3

1,020,294

1,039,699

19,405

0.8678

Yes

  

chr4

27,707,990

27,750,008

42,018

0.5614

No

  

chr4

41,465,452

41,496,569

31,117

0.8426

Yes

  

chr6

45,738,703

45,776,348

37,645

0.5049

Yes

  

chr6

89,209,799

89,220,599

10,800

0.6477

Yes

  

chr8

36,073,799

36,145,799

72,000

0.5122

Yes

  

chrX

37,290,568

37,303,155

12,587

0.8150

No

  

chrX

37,564,199

37,614,599

50,400

0.5427

No

  

chrX

56,120,456

56,149,298

28,842

0.6124

No

  

chrX

84,230,177

84,255,543

25,366

0.5679

No

  

chrX

138,374,999

138,386,999

12,000

0.5528

No

8

High3

chr13

48,993,325

49,013,328

20,003

0.5149

Yes

  

chr15

26,576,999

26,602,199

25,200

0.6022

Yes

  

chr25

32,374,157

32,403,637

29,480

-0.8278

Yes

  

chr4

33,564,599

33,578,999

14,400

0.5363

Yes

  

chr4

41,466,599

41,495,399

28,800

0.5097

Yes

  

chr6

45,744,065

45,772,999

28,934

0.5284

Yes

9

Low3

chr1

5,249,999

5,285,999

36,000

0.5559

Yes

  

chr1

144,107,850

144,130,905

23,055

0.5949

No

  

chr13

48,991,360

49,017,997

26,637

0.6031

Yes

  

chr17

73,139,605

73,159,081

19,476

-2.0603

No

  

chr18

6,080,815

6,121,152

40,337

-0.5749

No

  

chr25

32,362,844

32,470,747

107,903

0.6669

Yes

  

chr29

19,412,812

19,444,215

31,403

0.6690

Yes

  

chr3

1,020,599

1,038,599

18,000

0.6787

Yes

  

chr4

33,564,599

33,578,999

14,400

0.6100

Yes

  

chr4

41,465,452

41,487,890

22,438

0.6229

Yes

  

chr6

89,208,198

89,218,288

10,090

0.6937

Yes

  

chr8

36,077,399

36,152,999

75,600

0.5089

Yes

  

chrU

12,620,478

12,665,758

45,280

0.7441

No

10

Self4

chr13

48,992,999

49,010,999

18,000

0.5729

Yes

Chr: chromosome, Log R: log2Ratio, Shared: Yes/No - events shared among samples (i.e. hybridizations) or not.

Figure 2

False positive event calls could be due to high GC content. A 5.5 kb variable region (chr25:28829889-28835660) was identified in one control self to self array CGH. GC Percent in 5-Base Windows, Array CGH probe, Gap, RefSeq Gene and Repeat tracks are displayed in Btau_4.0. The GC percent track shows the percentage of G (guanine) and C (cytosine) bases in 5-base windows. The horizontal line at 41.7 in GC percent track represents the genome average of GC%. Probe locations are labeled like CHR25FS027220642 and etc.

In this project, we employed array CGH to study genomic integrity in cattle transgenic cell lines. This high-resolution genome-wide survey fills the knowledge gaps left out in the existing literature. Our results generate a valuable tool for genomic integrity evaluation and largely exclude the occurrences of large genomic structural variations (≥ 10 kb) during animal cloning, supporting our recent findings that epigenetic errors introduced by multiple rounds of cloning and/or genetic targeting are the possible underlying causes for the cloning efficiency decline [8, 9]. However, this initial genomic integrity survey reported here is probably not complete as the CGH arrays were designed by using only one reference genome. As a result, sequences absent in Dominette and present in other animals cannot be ascertained. Also, array CGH cannot detect small event (<5 kb) and balanced events like inversions and translocations. Therefore, we cannot totally exclude the possibility that both genetic and epigenetic influences may be at work and genetic differences may have played a role in the low efficiencies. With the costs of genome sequencing dropping dramatically by using next-generation sequencing, emerging high-quality cattle genomic sequence will soon facilitate the application of the direct sequence comparison strategy. Furthermore, additional studies like epigenomics are warranted and may unravel the epigenetic basis for the successful and efficient animal cloning.

Declarations

Acknowledgements and Funding

We thank D. Hebert, A. Edwards and W. Gang for technical assistance.

Authors’ Affiliations

(1)
USDA-ARS, ANRI, Bovine Functional Genomics Laboratory
(2)
Department of Animal and Avian Sciences, University of Maryland
(3)
Hematech Inc
(4)

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Copyright

© Liu et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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