Transgenic Res DOI 10.1007/s11248-014-9834-8

ORIGINAL PAPER

Efficient creation of an APOE knockout rabbit Diana Ji • Guojun Zhao • Allison Songstad Xiaoxia Cui • Edward J. Weinstein

•

Received: 27 March 2014 / Accepted: 4 September 2014 Ó Springer International Publishing Switzerland 2014

Abstract The rabbit is a preferred model system for diverse areas of human disease research, such as hypertension and atherosclerosis, for its close resemblance to human physiology. Its larger size than that of rodents allows for more convenient physiological and surgical manipulations as well as imaging. The rapid development of nuclease technologies enables the rabbit genome to be engineered as readily as that of rats and mice, offering rabbit models a chance to make their due impact on medical research. Here, we report the efficient creation of an APOE knockout rabbit by using zinc finger nucleases. The knockout rabbits had drastically elevated cholesterol and moderately increased triglyceride levels, mimicking symptoms in human heart disease. So far the rabbit genome has been successfully modified with three nuclease technologies. With a gestation period only days longer than those of rodents, we hope additional reports on their creation and characterization will help encourage the use of rabbit models where they are most relevant to human conditions. Keywords Gene targeting  Knockout rabbit  Animal model  APOE  Zinc finger nuclease (ZFN)

D. Ji  G. Zhao  A. Songstad  X. Cui  E. J. Weinstein (&) SAGE Labs, St. Louis, MO, USA e-mail: [email protected]

Introduction Rabbits are important laboratory animals for use as models in studying atherosclerosis, thrombosis, and ocular disease (Brousseau and Hoeg 1999), as well as embryonic development (Duranthon et al. 2012). However, being the only organism where gene targeting is available in embryonic stem cells, genetically engineered mice have been the dominant model system in the biomedical landscape for over 20 years, even for diseases in which the mouse model manifests little human clinical presentation (Rohra and Qazi 2008). The advantages of using a rabbit to model certain human disease have been clearly demonstrated (Yanni 2004; Pogwizd and Bers 2008). For example, the rabbit heart is large enough to allow for surgical and catheter-based interventions in a much easier manner than microsurgical approaches required of rodents, such as implantation of instrumentation for in vivo cardiac mapping studies. A rabbit can allow for larger amounts of tissue sampling for analysis. Similarly, imaging in the rabbit can achieve higher resolution. Finally, the rabbit’s cardiac physiology has greater similarity to that of humans than either the rat or mouse. For instance, rabbit ventricles are similar to humans and their lipoprotein metabolism is reflective of humans (predominantly LDL), but very different from mice (predominantly HDL) (Fan and Watanabe 2003). Historically, due to lack of tools for its genome engineering, the rabbit did not reach its full potential

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as a model system. Only transgenes were available in rabbits until recently, thanks to the rapid development of multiple nuclease technologies in the past few years that revolutionized the field of genetic engineering. Targeted modifications no longer have to rely on the availability of germline-competent embryonic stem cell lines in a given species. The first developed was zinc finger nuclease (ZFN) technology. Zinc finger nucleases (ZFNs) are a fusion between DNA-binding zinc finger proteins and the nuclease domain of restriction enzyme FokI (Kim et al. 1996; Smith et al. 1999). ZFNs can be designed to bind and cleave specific DNA sequences. The resulting double strand breaks are repaired in the cell through either highfidelity homologous recombination (HR) or the errorprone nonhomologous end joining (NHEJ) pathway (Lieber 1999; Pardo et al. 2009), often leading to nonsense mediated decay of targeted mRNA (Chang et al. 2007). ZFNs were used successfully to disrupt genes in many species, such as plants (Shukla et al. 2009), fruit flies (Bibikova et al. 2002), cultured mammalian cells (Porteus and Baltimore 2003; Santiago et al. 2008), zebrafish (Doyon et al. 2008), rats (Geurts et al. 2009; Mashimo et al. 2010), and mice (Carbery et al. 2010). The first rabbit knockout model was generated using ZFNs, disrupting the IgM gene (Flisikowska et al. 2011). Shortly after, the DNA binding domain of the transcription activator-like effector (TALE) was fused to the FokI nuclease domain and developed into TALE nucleases (TALENs), which like ZFNs, can be designed to cleave specific sequences. TALENs were also successfully used in creating gene disruptions in various species, including the rabbit (Song et al. 2013). More recently, the CRISPR/Cas9 system has been developed into an efficient targeting method at an explosive rate, owing to its ease to design and production (Jinek et al. 2012). Gene knockouts have been created in many species (Niu et al. 2014; Zhao et al. 2014; Ren et al. 2013; Auer et al. 2014; Wang et al. 2013), in some cases with multiple genes knocked out in the same animals, such as the disruption of four genes in the rabbit using the system (Yang et al. 2014). To expand our gene targeting capability using ZFNs, we decided to target a gene in the rabbit to create a relevant human disease model. The rabbit is the classically preferred animal for the study of diet induced atherosclerosis and has been found to exhibit

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hypercholesterolaemia within a few weeks of administration of high cholesterol diets. High cholesterol diets alone, however, fail to produce an optimal model. While short-term diet can induce early lesions similar to the fatty streaks observed in humans, longerterm studies are not practical due to associated hepatotoxicity (Yanni 2004). Apo-E is one of the best characterized apolipoproteins and plays critical roles in lipid transport, cholesterol homeostasis and health of the central nervous system (Hauser et al. 2011; Holtzman et al. 2012; Kanekiyo et al. 2014). The APOE gene has been disrupted in both mice (Plump et al. 1992) and rats (SAGE’s unpublished data: http:// www.sageresearchlabs.com/research-models/knockoutrats/apoe-knockout-rat), producing diverse and interesting phenotypes, relevant to various research areas, such as heart disease and Alzheimer’s disease. We believe that an APOE knockout rabbit would be a valuable addition to the existing atherosclerosis models. We hereby report the efficient disruption of the rabbit APOE gene via pronuclear microinjection of ZFNs. The homozygous null animals lacked apo-E protein and had elevated cholesterol and triglyceride levels, as expected. Whereas the model itself obviously needs more indepth characterization, the fact that genetic manipulation consistently works so efficiently should encourage researchers to consider rabbit models that best suit their research.

Materials and methods ZFN validation in cultured cells ZFN plasmids were designed and assembled by the Sigma CompoZr product line. SIRC cells were grown in DMEM with 10 % FBS at 37 °C with 5 % CO2. ZFN plasmid DNA was paired at a 1:1 ratio and transfected into the SIRC cells using a Nucleofector (Lonza, Basel, Switzerland), following the manufacturer’s 96-well shuttle protocol. Culture media was removed 18 h post transfection and cells were incubated with 15 ll of trypsin per well for 5 min at 37 °C. Cell suspensions were then transferred to 100 ll of QuickExtract solution (Epicentre) and incubated for 10 min at 68 °C and 3 min at 98 °C. Extracted DNA was then used as a template in PCR reactions to amplify the target site region with the following

Transgenic Res

primer set: Cel-1 F2: 50 - gttctgagccgctgtttctc; Cel-1 R2: 50 - ctcctgcacctgatcagaca. All PCR reactions contain 1 ll of template DNA, 5 ll of buffer II, 5 ll of 10 lM each primer, 0.5 ll AccuPrime High Fidelity (Life Technologies, Carlsbad, CA) and water to 50 ll. PCR cycling conditions were: 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 60 °C for 30 s, and 68 °C for 45 s, with an extension of 68 °C for 5 min and a 4 °C hold. Three microliters of the above PCR reaction was mixed with 7 ll of 1 9 buffer II and incubated under the following program: 95 °C, 10 min, 95–85 °C, at -2 °C/s, 85–25 °C, at -0.1 °C/s, 4 °C forever. One microliter each of nuclease S (Cel-I) and enhancer (Transgenomic, Omaha, NE) were added to digest the above reaction at 42 °C for 20 min. The mixture was resolved on a 10 % polyacrylamide TBE gel (BioRad, Hercules, CA). ZFN mRNA purification. The plasmids were linearized at the XbaI site, found 30 of the FokI endonuclease open reading frame. The MessageMax T7 capped transcription kit and poly (A) polymerase tailing kit (Epicentre, Madison, WI) were used to prepare 50 capped and 30 polyA tailed message RNA. Prepared mRNA was double precipitated with an equal volume of 5 M NH4OAc and dissolved in injection buffer (1 mM Tris–HCl, pH 7.4, 0.25 mM EDTA) and concentration was determined with a NanoDrop 2000 Spectrometer (Thermo Scientific, Wilmington, DE).

Rabbit oocyte microinjection and animal husbandry NZW rabbits were housed in the animal facility of Auricoop (ImmunoGene, Hungary), which operates under the supervision of Auricoop’s IACUC. Female donor New Zealand White (NZW) rabbits were injected with 120 U of pregnant mare’s serum gonadotropin and 150 U of human chorionic gonadotropin in order to induce superovulation. The rabbits were mated with a fertile male on day 4 post gonadotropin administration. Fertilized eggs were harvested for pronuclear microinjection with validated ZFN pair 11/13 (5 ng/ll) immediately following the collection. The injected eggs were incubated for 1 h following microinjection and transplanted into the oviducts of pseudopregnant recipient rabbits under anesthesia.

Founder identification Ear biopsies were incubated in 100 ll of QuickExtract (Epicentre Biotechnology) at 50 °C for 30 min, 65 °C for 10 min and 98 °C for 3 min. The extracted DNA was amplified with PlatinumÒ Pfx DNA polymerase (#11708, Life Technologies) and 5 % DMSO (Sigma). Each PCR reaction was cloned using TOPO TA cloning kit (Invitrogen) following the manufacturer’s instructions. At least 12 colonies were picked from each transformation, PCR amplified with T3 and T7 primers, and sequenced with either T3 or T7 primer. Sequencing was performed at Elim Biopharmaceuticals (Hayward, CA). Primers used for genotyping are following: 350 lg del F: 50 - tccgctgttgattgacagtt; 350 lg del R: 50 - gagaggtcaaaccggagt; lg del F: 50 - agagaacg tggtggcctgt; lg del R: 50 - tcctccagctccgacttgta. Plasma analysis Approximately 500 ll of plasma was collected from each rabbit after an overnight fast in heparin tubes and submitted for full blood chemistry analysis (Comparative Clinical Pathology Services, LLC, Columbia, MO). Each cohort ranged from 2 to 7 rabbits between the ages of 3–4 months. Protein analysis Liver and brain tissues from a 3-month old rabbit homozygous for an 8 bp deletion in the APOE gene and an age-matched wild type control were homogenized in lysis buffer containing 1 % SDS, 8 M urea, 50 mM Tris–HCl, pH 6.8, 2 mM EDTA, 2 mM DTT and 1X protease inhibitor cocktail (Sigma-Aldrich) (Zhao et al. 2010). The protein concentration of the lysate was normalized with the BCA kit (Thermo Fisher). Approximately 30 lg of total protein per sample was resolved on a 4–20 % SDS–PAGE and transferred onto nitrocellular membrane (Bio-Rad). The membrane was probed first with goat anti-rabbit apo-E antibodies from antibodies-online-Gmbh (Aachen, Germany, catalog number: ABIN236819, immunogen: rabbit apo-E protein) or Genway Biotech (San Diego, USA, catalog number: 11-511-248656, antigen: rabbit apo-E protein), and then with HRP- conjugated anti- goat secondary antibody (Abcam). The same membrane was reprobed with anti-beta actin antibody (Sigma-Aldrich). Western blot images were captured by Chemidoc XR ? (Bio-

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(A) bp

ZFN Pairs

ZFN Pairs M G

11 13

12 13

14 16 15 17

bp

500

M

G

21 23

22 23

24 25

24 26

27 28

29 31 30 33

500

300

316

150

181 135

369

300

237

50

(B)

150

132

50

E1

E2

E3

E4

Fig. 1 Zinc finger nuclease targeting and validation. a In vitro ZFN validation in SIRC cell line using Cel-1 mutation detection assay. Out of 11 pairs screened, 4 pairs target exon 3, and 7 pairs target exon 4. Active pairs are boxed. The expected sizes of each PCR amplicon and cleaved products are labeled in base pairs. M,

DNA size marker; G, GFP transfection control; ZFN pairs are labeled with design numbers. b The ZFN target sequence for pair 11/13 is shown, with underlined lowercase sequence indicating binding site and uppercase sequence indicating FokI cleavage site

Rad) using SuperSignal West Dura Chemiluminescent Substrate (Pierce).

Table 1 Injection statistics

Results ZFN design and microinjection

Session 1

2

3

4

Embryos collected

207

119

120

111

557

Embryos injected

196

98

96

75

465

Embryos transferred

122

76

68

60

326

9

1

2

4

16

Kits born

To obtain ZFNs specifically targeting the rabbit APOE gene, we first verified the APOE gene sequence in the New Zealand White (NZW) rabbit background because the genome of the NZW rabbit is not fully sequenced. We PCR amplified and sequenced the APOE gene from the SIRC rabbit cell line, derived from the cornea of the NZW strain. ZFNs were then designed to target the first two-thirds of the open reading frame. Sixteen pairs of ZFNs were assembled as previously described (Carbery et al. 2010) and transfected into SIRC cells to validate activity. Out of the 16 pairs screened, two pairs exhibited noticeable cleavage activity, pair 11/13 and pair 24/26 (Fig. 1a). The banding pattern of pair 27/28 was independent of endonuclease treatment, likely caused by nonspecific amplification. Pair 27/28 was not considered further.

123

Total

Pair 11/13, which targets APOE exon 3, was chosen to be used for the generation of the knockout rabbit (Fig. 1b). In vitro transcripts of ZFNs 11 and 13 were prepared as described previously and paired at a 1:1 ratio to a final concentration of 5 ng/ll for pronuclear microinjection. Single-cell rabbit embryos were harvested from superovulated females 1 day post mating, injected and transplanted into pseudopregnant females to obtain mutant founders (Bodo et al. 2004). Due to the low survival rate of microinjected embryos, a significant number was injected in order to generate sufficient numbers of live kits (Table 1). Sixteen total live kits were born from the 465 embryos microinjected and implanted.

Transgenic Res

(A)

Intron 2

Exon 3

WT Δ8 (#5, #14) Δ17 (#5) Δ6 (#11) Δ7+3 (#12) Δ8+22 (#12, #14)

(B) WT:

Δ8: Δ17:

(C) kDa

WT

Liver KO

WT

Brain KO

40 apo-E 30

β-Actin

Fig. 2 Genotype and protein expression of APOE knockout founders. b Mutant alleles from four out of five founders were aligned with the wild type sequences. ZFN binding sites are underlined. A dash represents a deleted nucleotide. Inserted sequences are in bold letters. The intron 2/exon 3 boundary is labeled with a dashed vertical line. D8, D17, and D6 refer to 8 bp, 17 bp and 6 bp deletion, respectively. D8 ? 22 and D7 ? 3 refer to 8 bp deletion with a 22 bp insertion and a 7 bp deletion with a

Identification of APOE knockout founders To identify ZFN-mediated mutations in the 16 live births, genomic DNA was extracted from ear clips, and the region immediately flanking the target site was PCR amplified and TOPO TA cloned and sequenced. Five of the 16 kits contained mutant alleles in the APOE gene, resulting from NHEJ-mediated repair of the ZFN cleavage at the target site. Notably, four founders did not contain wild type APOE sequence and were biallelically disrupted. With the exception of a 294 bp deletion identified in founder #1, the remaining mutations identified are small deletions ranging from 4 to 17 bp. Shown in Fig. 2a is the aligned mutant sequences, except from that of founder #1. Founder #5 has two different mutations, an 8 bp deletion and a 17 bp deletion. Interestingly, founder #14 has an allele with the same 8 bp deletion as in founder #5 and another with the 8 bp deletion as well as a 22 bp insertion 26 bp into intron 2. The 8 bp

3 bp insertion, respectively. Founder IDs are in parentheses after each genotype. b The predicted partial protein translations from two mutations. The first premature stop codon is indicated with an asterisk (*). c The 35 kDa band of rabbit apo-E is apparent in the liver and brain of wild type rabbits but not from the homozygous knockout rabbits after probing with anti-rabbit apo-E antibody. The same membrane was reprobed with anti-beta actin antibody

deletion in founders #5 and #14 and the 17 bp deletion in founder #5 are in exon 3 and lead to framshift and premature stop codons downstream (Fig. 2b). Germline transmission To test germline transmission of these mutations, founders #5 and #14 were mated to wild type NZW rabbits to generate F1 heterzoygous animals. Both founders produced four healthy litters, and all kits were heterozygous for one of the mutant alleles (Table 2), demonstrating 100 % germline transmission of all detected mutations and also further confirming that both founders were compound heterozygotes. Loss of protein in APOE homozygous null rabbits Apo-E protein is highly expressed in the liver and the brain. To examine whether the frameshift deletions in

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Transgenic Res Table 2 Germline transmission of two founder APOE knockout rabbits Female

Male

Litter

Mutation

# of kits/total

#5

WT

1

D8

3/6

D17

3/6

D8

3/3

D17

0

D8

4/9

D17

5/9

D8

3/8

D17

5/8

2 3 4 WT

#14

1 2 3 4

D8

5/10

D8 ? 22

5/10

D8

1/9

D8 ? 22

8/9

D8

1/1

D8 ? 22

0

D8 D8 ? 22

1/3 2/3

the gene resulted in the loss of apo-E protein, western blot analysis was performed using tissues from a 3-month old homozygous null rabbit containing the 8 bp deletion and an age-matched wild type control with goat anti-rabbit apo-E antibody (Antibodiesonline, Gmbh). The expected 35 kDa rabbit apo-E protein was present in the wild type liver and brain tissues, but was absent in both tissues of the knockout (Fig. 2c), confirming loss of protein in the knockout rabbits. Similar results were observed with another anti-rabbit apo-E antibody from another commercial source (Genway Biotech, data not shown). Interestingly, apo-E from the rabbit brain had a slightly higher molecular weight than that from the liver on SDS– PAGE, in agreement with previously reported differential posttranslational modifications in the two tissue types (Huang et al. 2013; Reardon et al. 1986). Blood chemistry Apo-E is known for its role in lipid transportation. apoE deficiency leads to elevated plasma cholesterol in mouse (Plump et al. 1992; Zhang et al. 1992) and rat models (www.sageresearchmodels.com). To investigate the effect of apo-E deficiency in rabbits, we analyzed the plasma from wild type New Zealand White rabbits, rabbits with a homozygous deletion of

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APOE, and those with a heterozygous APOE geneotype. All were fed a standard diet, and blood was drawn at 4–5 months of age. As expected, the plasma cholesterol levels of the homozygous null rabbits are 2–5 times higher than those of the age-matched wild type controls (Table 3). Elevated levels of triglycerides were also observed in the founders (data not shown) and homozygous null rabbits, although LDL and HDL levels remain unchanged. Compared to its wild type counterparts, APOE deficient rabbits show higher plasma levels of alanine aminotransaminase (ALT) and alkaline phosphatase (ALP), both biomarkers for liver injury.

Discussion In addition to being a classical experimental model system, the rabbit also has the advantage of a short gestation period (30–31 days) and large litters. However, due to limited tools and techniques available in the precise manipulation of the rabbit genome, it remains an infrequently utilized disease model organism, and is rather more often used for antibody production. The first knockout rabbit was created via pronuclear microinjection of ZFNs, where the IgM locus was disrupted (Flisikowska et al. 2011). Another immunodeficient rabbit has been generated by the disruption of Rag1 and Rag2 using TALENs (Song et al. 2013). There is also proof-of-principle of using Cas9 to target multiple genes successfully (Yang et al. 2014). Here we present the first genetically engineered rabbit disease model with expected phenotype, an APOE knockout rabbit. We obtained a 31 % founder rate among 16 total live births screened, which is comparable to the efficiency rates of the IgM KO rabbit model (Flisikowska et al. 2011). Whereas rabbit embryos appear to be more sensitive than rodent embryos to manipulations during microinjection, requiring significant numbers of fertilized eggs to be harvested, injected, and implanted into surrogate females, the targeting rate observed is similar to that seen in rats and mice. Out of the five founders identified, four founders carried only mutant APOE alleles and were compound heterozygotes. Both founders that were bred to the F1 heterozygous generation produced normal sized litters with 100 % germline transmission of all identified alleles. Western analysis on brain and liver tissues

7

Gamma-glutamyltransferase (U/L)

9

40

28

8

1.1 0.1

51

7.8

45

145

13.4

17.9

26

100

3.9

140

2.9

1.4

4.1

73

5.5 52

F

4 month

Wildtype

8,521

29

25

21

5

1 0.2

73

8.9

176

109

13.6

18.2

30

97

4.2

141

2.0

2.1

4.1

45

6.2 103

M

4 month

(-/-)

9,957

25

50

19

5

0.8 0.1

51

9.7

212

428

13.6

15.8

28

99

3.8

139

2.3

1.8

4.2

74

6 68

M

4 month

(-/-)

9,961

20

41

17

3

0.7 0.1

66

8.1

162

189

13.7

20.9

26

97

3.9

140

2.4

1.8

4.3

66

6.1 78

M

5 month

(-/-)

9,962

15

34

23

5

0.7 0.1

56

9

139

149

13.2

21.8

23

99

3.8

140

2.2

1.9

4.2

55

6.1 75

M

4 mon

(-/-)

9,964

26

39

17

5

0.8 0.1

48

9.6

183

184

13.3

20.9

26

96

3.9

139

2.3

1.9

4.3

76

6.2 94

M

4 month

(-/-)

9,965

27

40

18

6

1 0.2

68

11.6

217

123

13.7

32.2

19

98

5.2

144

1.8

2.2

4

87

6.2 69

F

4 month

(-/-)

9,966

13

62

21

6

1 0.2

55

9.6

68

149

13.5

21.9

29

95

3.9

142

2.1

2

4.1

52

6.1 77

M

5 month

(-/?)

9,958

9

57

21

6

1 0.1

56

8.9

62

138

13.3

19.4

26

98

4.4

139

2.1

2

4.1

59

6.1 74

M

5 month

(-/?)

9,959

32

53

18

3

0.7 0.1

51

9.4

87

146

13.4

20.5

26

96

3.5

139

2.4

1.8

4.3

91

6.1 136

M

4 month

(-/?)

9,963

Wild type, homozygous APOE knockout rabbits, and heterozygous APOE knockout rabbits were subjected to a blood draw at 4–5 months of age

31

1 0.1

Creatinine (mg/dl) Total billirubin (mg/dl)

LDL (mg/dl)

41

Alanine aminotransferase (U/L)

24

7.7

Phosphorus (mg/dl)

56

86

Cholesterol (mg/dl)

HDL (mg/dl)

97

Alkaline phosphatase (U/L)

Blood urea nitrogen (mg/dl)

13.4

4.1

Potassium (mEq/L)

19.1

141

Sodium (mEq/L)

Calcium (g/dl)

2.6

Albumin/globulin

Anion gap

1.6

Globulin (g/dl)

99

4.2

Albumin (g/dl)

27

59

Glucose (mg/dl)

Total CO2 (mEq/L)

5.8 54

Total protein (g/dl) Triglycerides (mg/dl)

Chlorid (mEq/L)

4 month F

Gender

Wildtype

APOE genotype

Age at time of sampling

8,520

Animal ID

Table 3 Blood chemistry

25

49

18

6

0.9 0.1

49

9

72

130

13.1

23

26

95

4

140

2.7

1.5

4

87

5.5 73

F

4 month

(-/?)

9,967

21

40

19

4

1 0.1

53

8.8

192

179

13

20.2

26

98

4.2

140

2.2

1.8

4

69

5.8 79

F

4 month

(-/?)

9,968

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confirmed the lack of apo-E expression in F2 homozygous knockout animals. APOE knockout mice were reported to have higher cholesterol than their wild type littermates and display a host of phenotypes associated with hyperlipidemia, including and the presence of fatty streaks and preatherosclerotic lesions in the proximal aorta (Plump et al. 1992; Zhang et al. 1992). The APOE KO rabbits we present here displayed a similar adverse lipid profile, with elevated cholesterol levels that are 2–5 times higher than wild type, as well as increased levels of triglycerides, alanine aminotransaminase, and alkaline phosphatase, indicating possible liver injury, as observed in APOE KO mice (Ferre et al. 2009). Given the rabbit’s close resemblance of human physiology, this model should be useful for novel and improved approaches in understanding the mechanisms of hypercholesterolemia and the role of apo-E in lesion development upon a more in-depth characterization. With the rapid development of the nuclease technologies, genome engineering becomes more feasible for many species that were not possible before. The rabbit has been knocked out using all three technologies and shown consistent high efficiencies, proving that the rabbit genome can be reliably modified. The decision to switch from a widely-accepted model organism, such as the mouse, to one less used in the field is not trivial, even when there are clear advantages in the new model organism. However, oftentimes reluctance is due to a mere lack of information. We hope the continuous reports on the creation and characterization of genetically modified rabbits will provide the necessary confidence in researchers to choose the rabbit as the model system when their research can most benefit. Acknowledgments We would like to thank Eliezer Kopf and Fishel Alon for housing, breeding, and tissue sampling for genotyping and serum analysis, and Zsuzsanna Bosze for embryo microinjection service.

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