This work was partially funded by grants from the National Institutes of Health (HL105114, HL088391, NS066652, and HL068878 to Y.E.C), the American Heart Association (National Scientist Development 0835237N to J.Z.). Y.E.C. is financially supported as endowed Frederick Huetwell Professor of Cardiovascular Medicine at the University of Michigan Medical Center (UMMC). This work utilized Core Services supported by Center for Advanced Models for Translational Sciences and Therapeutics (CAMTraST) at UMMC.

All animal maintenance, care and use procedures were reviewed and approved by the University Committee on the Use and Care of Animals of the University of Michigan.
1. ZFN Design and Selection
<ol>
	<li>Supply the genomic sequence information of the gene of interest (e.g. rabbit ApoCIII) to the manufacturer. Select ZFN set(s) for the experiments based on the ZFN scores.</li>
</ol>
2. Rabbit Embryo Collection
<ol>
	<li>Superovulate sexually matured (6-18 months) New Zealand White (NZW) female rabbits by subcutaneous (s.c.) injection of FSH twice/day with a dosage of 3 mg for the first two injections, 5 mg for the next two injections and 6 mg for the last two injections.</li>
	<li>Administer a single intravenous (i.v.) injection of 200 IU hCG at 72 hr after the first FSH injection to induce ovulation. Mate the superovulated females with a male rabbit immediately after hCG injection.</li>
	<li>Euthanize the superovulated donor rabbits with sodium pentobarbital (150 mg/kg, i.v.) at 16-18 hr&#160;post insemination (p.s.i.).</li>
	<li>Recover the oviduct ampullae by carefully collecting the entire oviducts and ovaries structure.</li>
	<li>Flush each oviduct with 10 ml manipulation medium of HEPES buffered TCM 199 supplemented with 10% fetal bovine serum, by injecting the medium from the uterus end of the oviduct. Collect the flushed medium at the ovary-end opening of the oviduct to a Petri dish.</li>
	<li>Observe and identify recovered embryos in the flushed medium, wash the embryos three times in the manipulation medium, keep them in the manipulation medium at 38.5 &#176;C in air, and observe them under a stereomicroscope for the occurrence of fertilization. Prepare for microinjection on pronuclear stage embryos 19-21 h p.s.i.</li>
</ol>
3. Preparation of ZFN mRNA
<ol>
	<li>Purify the resulting mRNA before resuspension in RNAse-free 0.1X TE (1 mM Tris-Cl pH 8.0, 0.1 mM EDTA). Measure the concentration and store the mRNA at -80&#176;C until use.</li>
	<li>Prepare the mRNAs encoding ZFN pair (4-10 ng/&#956;l in 1 mMTris-Cl, 0.1 mM EDTA at pH 7.5), aliquot into 10&#160;&#956;l vials. Store them at -80 &#186;C until ready for use. Before microinjection, thaw the RNA vial(s), and keep it on ice.</li>
</ol>
4. Embryo Microinjection
<ol>
	<li>Prepare the microinjection platform by placing a 5 &#956;l drop of manipulation medium on a 1-well chamber slide that has the media chamber removed. Cover the medium drop with mineral oil and place on the heated microscope stage.</li>
	<li>Place holding pipette (120&#160;&#956;m diameter) into the holder arm of the micromanipulator and position it into the drop of manipulation medium.</li>
	<li>Fabricate injection micropipettes by heating and pulling borosilicate glass capillary tubes in a micropipette puller device. Load the mRNA to be injected in the injection pipette.</li>
	<li>Turn on the compressed air supply that is connected to a microinjector.</li>
	<li>Place injection pipette into the injection holder, put it on the micromanipulator and position it into the drop of manipulation medium.</li>
	<li>Setup the microinjector, with the following suggested parameters: holdP=2 p.s.i., InjP=20 p.s.i., ClearP= 60 p.s.i., Injtime= 1 sec, which will result in an injection volume of 1-5 pl.</li>
	<li>Open the tip of the injection pipette by gently tapping it against the holding pipette.</li>
	<li>Transfer embryos (30-50) to the micromanipulation drop on the platform. Use the holding pipette to capture an embryo and align the injection needle, embryo and the holding pipette along the x-axis.</li>
	<li>Under 400X magnification, advance the injection pipette through the embryo. Be careful to avoid the nucleus. Once the plasma membrane is pierced, press foot pedal to inject. After injection, withdraw the needle, release the embryo. Repeat the process for all remaining embryos.</li>
	<li>Wash injected embryos three times in embryo culture medium, which consists of Earle&#39;s Balanced Salt Solution (EBSS) supplemented with nonessential amino acids (NEAA), essential amino acids (EAA), 1 mM L-glutamine, 0.4 mM sodium pyruvate, and 10% FBS.</li>
	<li>Incubate embryos at 38.5 &#176;C in 5% CO2 in air for 1-2 hr before they are surgically transferred into the oviducts of a synchronized recipient female NZW rabbit (see step 5.1). Alternatively, culture the embryos until they reach blastocyst stage before they are used for in vitro validation or assays.</li>
</ol>
5. Embryo Transfer
<ol>
	<li>Select a female NZW rabbit 5-12 months old, in good health, weighing approx. 4.0 to 5.0 kg as recipient animal. Administer Gonadotropin Releasing Hormone (GnRH) injection intramuscularly (i.m., 50 mcg/ml, 0.3 ml/animal) to the recipient at the same time point with that of hCG injection to the donor animal(s) (see step 2.2).</li>
	<li>After completion of microinjection, prepare the recipient rabbit for embryo transfer (normally 16-24 hr after GnRH injection). Anesthetize the rabbit with 10-40 mg/kg ketamine (i.m.), and/or vaporized isoflurane (2-4%) through inhalation. Protect the rabbit&#8217;s eyes from excessive drying by the application of a sterile ophthalmic ointment. Observe the animal until it is fully unconscious, i.e. does not respond to pedal reflexes; squeezing the foot pad is used to check for lack of pedal reflex which is an indication of adequate anesthesia.</li>
	<li>Shave the rabbit&#39;s abdomen, wash with antiseptic scrub, followed by antiseptic solution, and wipe the shaved area with sterile gauze sponges.</li>
	<li>Perform surgery under sterile conditions, record vital signs of the recipient rabbit approximately every 15 min throughout the surgery.</li>
	<li>Transfer 10-25 embryos to one recipient animal, depending on the available number of embryos.</li>
	<li>At the end of the surgical procedures, close the muscle layer with coated absorbable suture. Suture the skin with either a subcuticular suture using absorbable suture or with simple interrupted skin sutures using nonabsorable suture (e.g. monofilament nylon or similar).</li>
	<li>Keep the animal warm and monitor it until its sternal recumbency is attained.</li>
	<li>Monitor the animal daily. Follow veterinarian&#8217;s instructions to care for the animal. Administer meloxicam (s.c. 1.0 mg/kg) to relieve pain and/or reduce fever daily post operation if determined necessary by the veterinarian.</li>
	<li>Remove nonabsorable skin sutures at about 10-14 days post operation.</li>
</ol>
6. Detection of ZFN Induced Gene Disruption
<ol>
	<li>For in vitro validation, perform genotyping on the in vitro cultured embryos. Lyse individual embryos that developed to morula/blastocyst stages in 5 &#956;l NP40 solution (1% NP40 plus 1 ug/ml Proteinase K in Taq Buffer) for 0.5-1 hr at 55 &#176;C, and 10 min at 95 &#176;C.</li>
	<li>For genotyping of the offspring, collect ear skin samples from newborn kits, extract genomic DNA, and use them as templates for PCR sequencing. Apply Kwik-Stop to the sampling area after collection to stop bleeding and reduce discomfort.</li>
	<li>Use the lysis from step 6.2 or 6.3 as template for PCR using LA-Taq and PCR primer pair, which are located upstream (5&#8217;- TGAGGCCGGGAAGGGAGCAGTCG-3&#8217;) and downstream (5&#8217;-GCCAGGCCCACCCACGGAACAGC-3&#8217;) of the ZFN target site.</li>
	<li>Purify PCR products and sequence them with sequencing primer (5&#8217;-TCTGCACGCTTGGGGCTGGAG-3&#8217;).</li>
	<li>Identify mutant samples by looking for double curve in the sequence diagram around the targeting site. Label the ones with double curves as &#34;positive&#34;.</li>
	<li>Clone the &#34;positive&#34; PCR products into TA cloning vector, pick up 10-20 clones, sequence the inserts, and confirm the mutations around the targeting site.</li>
</ol>

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	<li>Ooi, E. M., Barrett, P. H., Chan, D. C., Watts, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+C-III:+understanding+an+emerging+cardiovascular+risk+factor.">Apolipoprotein C-III: understanding an emerging cardiovascular risk factor.</a> Clinical Science. 114, 611-624 (2008).</li><li>Pollin, T. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+null+mutation+in+human+APOC3+confers+a+favorable+plasma+lipid+profile+and+apparent+cardioprotection.">A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection.</a> Science. 322, 1702-1705 (2008).</li><li>Ginsberg, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+B+metabolism+in+subjects+with+deficiency+of+apolipoproteins+CIII+and+AI.+Evidence+that+apolipoprotein+CIII+inhibits+catabolism+of+triglyceride-rich+lipoproteins+by+lipoprotein+lipase+in+vivo.">Apolipoprotein B metabolism in subjects with deficiency of apolipoproteins CIII and AI. Evidence that apolipoprotein CIII inhibits catabolism of triglyceride-rich lipoproteins by lipoprotein lipase in vivo.</a> The Journal of Clinical Investigation. 78, 1287-1295 (1986).</li><li>Cohn, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+apoC-III+production+is+a+characteristic+feature+of+patients+with+hypertriglyceridemia.">Increased apoC-III production is a characteristic feature of patients with hypertriglyceridemia.</a> Atherosclerosis. 177, 137-145 (2004).</li><li>Cohn, J. S., Patterson, B. W., Uffelman, K. D., Davignon, J., Steiner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rate+of+production+of+plasma+and+very-low-density+lipoprotein+(VLDL)+apolipoprotein+C-III+is+strongly+related+to+the+concentration+and+level+of+production+of+VLDL+triglyceride+in+male+subjects+with+different+body+weights+and+levels+of+insulin+sensitivity.">Rate of production of plasma and very-low-density lipoprotein (VLDL) apolipoprotein C-III is strongly related to the concentration and level of production of VLDL triglyceride in male subjects with different body weights and levels of insulin sensitivity.</a> The Journal of Clinical Endocrinology and Metabolism. 89, 3949-3955 (2004).</li><li>Maeda, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+apolipoprotein+C-III+gene+in+mice+results+in+hypotriglyceridemia+and+protection+from+postprandial+hypertriglyceridemia.">Targeted disruption of the apolipoprotein C-III gene in mice results in hypotriglyceridemia and protection from postprandial hypertriglyceridemia.</a> The Journal of Biological Chemistry. 269, 23610-23616 (1994).</li><li>Jong, M. 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The authors have nothing to disclose.

In the present work, five ApoCIII KO rabbits were generated using the ZFN technology. Previously the only report of the production of GTT rabbits also used the ZFN technology18. The present work confirmed that ZFN is useful to effectively target genes in rabbits.
In the present study, the transgenic rate (positive kits/total born) is23.8% (5/21), which is comparable to that of the previous ZFN report in rabbits (30.8%, 16/52)18, and those reported of using ZFN in other animal species, including zebra fish19, mice20, rats21 and pigs17. This rate is in fact higher than the rates of many conventional transgenic rabbit production studies, which normally fall in the range of 5-20%. For example, in an effort to product ApoCIII transgenic rabbits via conventional DNA microinjection, Ding et al. obtained 3 positive founders out of 54 pups born (5.6%)22.
Consistent with previous findings, the mutations at the targeting sites are variable, including deletions or insertions consisting different number of bases (ranging from 1-21 in the five KO rabbits). Based on the specific sequence information of the founder animals, it is predicted that at least four (i.e. those containing &#916;1, +1, +1, or &#916;20 mutations) would have function loss of ApoCIII. Animal R-16 with &#916;21 may not display functional loss, as this mutation is predicted to only cause loss of seven amino acids, but not a reading frame shift. Ultimately, phenotype assays are necessary to determine if these founder animals truly display the loss of ApoCIII functions.
None of the five KO rabbits generated in the present study contain biallelic modifications. Interestingly, this is also the case in the previous ZFN rabbit report. In contrast, when ZFNs targeting &#945;1,3-galactosyltransferase were applied to pig cells, the frequency of targeting a single allele ranged from 7-46%, with approximately one-third of mutations creating single-step biallelic knockouts 23. Consistent with this, when TALEN was applied to pig fibroblast cells, the biallelic modifications were found in approximately 15-40% of the total positive clones14. It is possible that the failure to generate biallelic KO rabbits in the present study may indicate a species difference (i.e. rabbits vs. pigs) for such capacity of nuclease based gene targeting. It is, however, more likely that this is simply because the number of KO rabbits generated in this project is relatively small. We believe that ZFN is capable of generating bialleic mutations in rabbits, which should be considered as another potential advantage of ZFN based GTT rabbit production. Further experiments are needed to confirm such capacity of ZFN in rabbits.
In conclusion, the present work demonstrates that ZFN based gene targeting approach is effective in producing KO rabbits. In particular, we generated five ApoCIII KO rabbits with a satisfactory transgenic rate of 23.8%. These animals are believed to provide more meaningful information about this protein&#8217;s role on lipid metabolisms in humans than the corresponding mouse models. We predict ZFN based gene targeting, as well as other nuclease based technologies such as TALEN and RGEN, in nonmurine animals will significantly facilitate the development of novel animal models to study various human diseases.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50957/50957fig1.jpg" width="500px" /> 
Figure 1. Flow chart of production of knockout rabbits using ZFN technology. Production of KO rabbits using ZFN technology starts with the selection of the gene of interest. The sequence information is provided, ZFN sets are designed, and subjected to yeast based in-house validation. At check point (CP) 1 (CP-1), only those sets passed in-house validation (50% or higher of the internal positive control) will be provided to the users for selection. If no sets pass CP-1, additional sequence may be needed for designing effective ZFN sets. An in vitro embryo validation is followed to ensure the selected ZFN set can induce mutations at selected sites (CP-2). ZFN set(s) will only be used if it passes CP-2 (&#62;=10% mutation rates in in vitro embryos). Failure at CP-2 will require a redesign of the ZFN sets. Embryo donors will be prepared and pronuclear stage embryos will be microinjected with the validated ZFN sets. These microinjected embryos will be transferred to synchronized embryo recipients. After one month gestation period, newborns will be genotyped (CP-3). If none of the newborns are positive, additional microinjection will be performed. It takes 2-3 months from start of the project to CP-2, assuming no failure during the process. It takes additional 2-3 months from CP-2 to CP-3. Therefore it is possible to generate a knockout rabbit in a 4-6 month time frame using the ZFN technology. <a href="http://www.jove.com/files/ftp_upload/50957/50957fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50957/50957fig2.jpg" width="500px" /> 
Figure 2. ZFN design. Three ZFN sets (Set 1, 2, and 3) were designed targeting different sequences on Exon 1 or Exon 2 of rabbit ApoCIII (A). All three sets were subjected to the yeast MEL-1 reporter assay to determine the ZFN activities. According to the manufacture&#8217;s protocols, ZFNs that show &#62;50% activities of that of the manufacture&#8217;s internal positive control are regarded as useful for in vitro and in vivo genome editing experiments. The ZFN activities were 224.2% for Set 1, 196.9% for Set 2 and 177.8% for Set 3 (B), therefore Set 1 was selected in the present study. <a href="http://www.jove.com/files/ftp_upload/50957/50957fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/50957/50957fig3.jpg" width="500px" /> 
Figure 3. In vitro embryo validation of ZFN. The mRNA (5 &#956;g/ml) of Set 1 was microinjected to the cytoplasm of 35 pronuclear stage rabbit embryos (A). Flushed embryos without microinjection treatment (n=31) were used as the control group. The BL development rate was 77.4% in the control group. In the microinjected group, the BL rate was 51.4%. Of 16 BLs subjected to PCR sequencing, 8 (50%) were identified as positive, indicating Set 1 is an effective ZFN set to induce mutations at the targeting site (B, apoc3wt.seq, black-boxed) of ApoCIII in rabbits. The BLs containing mutations are BL#apoc-3, 4, 5, 7, 11, 13, 15, and 17 (B, red underlined). The remaining BLs (#apoc-2, 6, 9, 10, 12, 14 and 16) do not have mutations at the targeting site. <a href="http://www.jove.com/files/ftp_upload/50957/50957fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/50957/50957fig4.jpg" width="500px" /> 
Figure 4. Production of ApoCIII KO rabbits. One hundred and forty-five embryos microinjected with ZFN Set 1 mRNAs were transferred to 7 pseudo-pregnant recipient rabbits (A). Freshly flushed embryos (n=75) without ZFN microinjection were transferred to recipients (n=6) as the control group. The term rate calculated as total term kits/total embryos in experiment group is 14.5% (21/145), whereas the rate is 29.3% (22/75) in the control group (A). Out of 21 kits born in the microinjected group, five (R1, R9, R11, R12 and R16) were identified as positive KO kits after PCR sequencing (B). The indels at the targeting site include two insertions (+1 for both R9 and R11) and three deletions (&#916;1, &#916; 20, &#916; 21 for R1, R12 and R16, respectively). <a href="http://www.jove.com/files/ftp_upload/50957/50957fig4highres.jpg" target="_blank">Click here to view larger image</a>.

Apolipoprotein (Apo) C-III (ApoCIII) is a small O-glycosylated secretory protein that is synthesized mainly in the liver and intestine. It resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins (HDL). ApoCIII has been recognized as a risk factor for cardiovascular disease1. Patients with hereditary deficiency of ApoCIII have low plasma triglyceride (TG) levels and reduced subclinical coronary artery atherosclerosis2,3 . Elevated plasma ApoCIII level, on the other hand, often correlates with insulin resistance, obesity, and hypertriglyceridemia (HTG)4,5 .
Gene knockout (KO) is a powerful means to study the function of a gene. Consistent with the observations in human mutant populations, knockout of the ApoCIII gene in mice led to a reduction of plasma TG and protection from postprandial HTG6. Such positive role of ApoCIII appeared to be independent of Apolipoprotein E (ApoE), as mice deficient of both ApoE and ApoCIII are also protected from postprandial hyperlipidemia7. Although these ApoCIII KO mouse models have provided invaluable information on the possible functions of ApoCIII in humans, it is noted that the metabolism of lipoprotein of mice is different from that of humans in many aspects. For example, mouse lacks plasma cholesteryl ester transfer protein (CETP), a major enzyme involved in the transportation of VLDL, LDL, and HDL8. Therefore, it is necessary to develop an appropriate animal model to further understand the physiological role of ApoCIII in vivo.
The rabbit is a classical model animal species9-11. It has a short gestation period (30-31 days), large litter size (4-12/litter) and can be housed conveniently in an indoor facility. Compared to mice, rabbits are phylogenetically closer to humans10. Importantly, like human but unlike mice, rabbits are LDL-rich mammals and have substantial levels of CETP10,12 . Furthermore, they are susceptible to cholesterol-rich diet-induced atherosclerosis with the lesions similar to those seen in human atherosclerosis13. For these reasons, we hypothesized that ApoCIII KO rabbits can serve as a better model than their mouse counterparts to investigate the roles of ApoCIIIplay in lipid metabolism and atherosclerosis in humans.
Production of gene targeted transgenic (GTT) rabbits has been a challenge. This is mainly due to the lack of germline transmitting embryonic stem cells (ESC), and the extremely low efficiency of somatic cell nuclear transfer (SCNT) in rabbits. ESC is the primary tool to generate GTT mice, whereas and SCNT has been successfully applied to generate KO animals in species lacking germline transmitting ESCs, including pigs, sheep and cattle, but not rabbits. Recently Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN)14, and RNA Guided Endonuclease (RGEN)15emerged as powerful means for genome editing. These nucleases, so called &#34;molecular scissors&#34;, are efficient in generating double-strand breaks (DSB) in the genome that can lead to a functional KO of the targeted gene or used to integrate a DNA sequence at a specific locus in the genome in a number of species16. In 2011, among the first adapting the ZFN technology, we generated peroxisome proliferator-activated receptor gamma (PPAR&#947;) KO porcine cells via this approach and successfully generated PPAR&#947; KO pigs after using these cells for SCNT17. In the same year, efficient immunoglobulin gene disruption and targeted replacement in rabbits also using ZFN was reported18.
In the present study, five ApoCIII KO rabbits were generated as confirmed by PCR sequencing, using ZFN Set 1 (see Figure 1. for flow chart illustration). These animals are novel resources to study the roles of ApoCIII in lipid metabolisms.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">APOC3-ZFN</td>
			<td style="width:276px;">Sigma Aldrich</td>
			<td style="width:136px;">CSTZFNY-1KT</td>
			<td style="width:167px;">Target Gene: APOC3, Lot Number: 12201118MN</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mMESSAGE kit</td>
			<td>Invitrogen</td>
			<td>AM1344M</td>
			<td>mRNA synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEGAclear Kit&#160;</td>
			<td>Invitrogen</td>
			<td style="width:136px;">AM1908M</td>
			<td>mRNA purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Follicle-stimulating hormone</td>
			<td>Bioniche Life Sciences</td>
			<td style="width:136px;">Folltropin-V</td>
			<td>Treating embryo donor rabbits</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human chorionic gonadotropin</td>
			<td>Intervet</td>
			<td style="width:136px;">Chorulon</td>
			<td>Treating embryo donor rabbits</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gonadotropin-releasing hormone</td>
			<td>Prospecbio</td>
			<td>HOR-255</td>
			<td>Treating embryo recipient rabbits</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Earle&#39;s Balanced Salt Solution (EBSS)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:136px;">SH30029.02</td>
			<td>Embryo culture, base medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM (nonessential amino acid)</td>
			<td style="width:276px;">Sigma Aldrich</td>
			<td style="width:136px;">M7145</td>
			<td>Embryo culture, supplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BME AMINO ACIDS solution</td>
			<td style="width:276px;">Sigma Aldrich</td>
			<td style="width:136px;">B6766</td>
			<td>Embryo culture, supplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamine</td>
			<td>Gibco</td>
			<td>25030-149</td>
			<td>Embryo culture, supplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyruvate&#160;</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Embryo culture, supplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td style="width:276px;">Sigma Aldrich</td>
			<td>12003C</td>
			<td>Embryo culture, supplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES buffered TCM 199&#160;</td>
			<td>Gibco&#160;</td>
			<td style="width:136px;">12350039</td>
			<td>Embryo manipulation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Incubator</td>
			<td style="width:276px;">Eppendorf&#160;</td>
			<td style="width:136px;">Galaxy 170</td>
			<td>Embryo culture equipment</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Micromanipulator&#160;</td>
			<td>Eppendorf&#160;</td>
			<td style="width:136px;">TransferMan NK 2</td>
			<td>Embryo manipulation equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette puller</td>
			<td>Sutter Instruments Inc.</td>
			<td style="width:136px;">P-1000</td>
			<td>Embryo manipulation equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microinjector&#160;</td>
			<td>Tritech Research</td>
			<td style="width:136px;">MINJ-D&#160;</td>
			<td>Embryo manipulation equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass capillary tubes&#160;</td>
			<td>World Precision Instruments, Inc.</td>
			<td style="width:136px;">TW100F-6</td>
			<td>Embryo manipulation supply</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Euthasol (pentobarbitol sodium)</td>
			<td style="width:276px;">Virbac AH, Inc.</td>
			<td style="width:136px;">ANADA#200-071</td>
			<td>Euthanization of embryo donor rabbits</td>
		</tr>
	</tbody>
</table>

production of apolipoprotein c-iii knockout rabbits using zinc finger nucleases

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins (HDL). It has been recognized that high levels of plasma ApoCIII constitutea risk factor for cardiovascular diseases (CVD). Elevated plasma ApoCIII level often correlates with insulin resistance, obesity, and hypertriglyceridemia. Invaluable knowledge on the roles of ApoCIIIin lipid metabolisms and CVD has been obtained from transgenic mouse models including ApoCIII knockout (KO) mice; however, it is noted that the metabolism of lipoprotein in mice is different from that of humans in many aspects. It is not known until now whether elevated plasma ApoCIII is directly atherogenic. We worked to develop ApoCIII KO rabbits in the present study based on the hypothesis that rabbits can serve as a reasonablemodelfor studying human lipid metabolism and atherosclerosis. Zinc finger nuclease (ZFN) sets targeting rabbit ApoCIIIgene were subjected to in vitro validation prior to embryo microinjection. The mRNA was injected to the cytoplasm of 35 rabbit pronuclear stage embryos, and evaluated the mutation rates at the blastocyst state. Of sixteen blastocysts that were assayed, a satisfactory 50% mutation rate (8/16) at the targeting site was achieved, supporting the use of Set 1 for in vivo experiments. Next, we microinjected 145 embryos with Set 1 mRNA, and transferred these embryos to 7 recipient rabbits. After 30 days gestation, 21 kits were born, out of which five were confirmed as ApoCIII KO rabbits after PCR sequencing assays. The KO animal rate (#KO kits/total born) was 23.8%. The overall production efficiency is 3.4% (5 kits/145 embryos transferred). The present work demonstrated that ZFN is a highly efficient method to produce KO rabbits. These ApoCIII KO rabbits are novel resources to study the roles of ApoCIII in lipid metabolisms.

In vitro validation of ZFN pairs 
Sixteen ZFN pairs were initially designed. The targeted disruption sequence of Set 1 is located at Exon 2 of rabbit ApoCIII (Figure 2A). Three pairs (Set 1, 2, and 3, Figure 2A) were selected and subjected to the yeast MEL-1 reporter assay to determine the ZFN activities (Figure 2B). Set 1 has a ZFN score of 224.2%, higher than those of Set 2 (196.9%) and Set 3 (177.8%) and much higher than the selection threshold (i.e. 50% of the internal positive control). Therefore Set 1 was selected for in vitro validation experiments.
A particular set needs to result in a positive rates in the embryos of 10% or higher to pass the in vitro embryo validation, based on the internal validation criteria. The mRNA (5 &#181;g/ml) of Set 1 was microinjected to the cytoplasm of 35 pronuclear stage rabbit embryos (Figure 3A). Flushed embryos without microinjection treatment (n=31) were used as the control group. Eighteen embryos of the microinjected group developed to BL stage on D5, of which sixteen were sequenced, and eight (BL#apoc-3, 4, 5, 7, 11, 13, 15, 17, Figure 3B, red underlined) displayed mutated sequences at the targeting site (black-boxed, Figure 2B, top row, apoC3wt.seq). The BL rate of the microinjected group (51.4%) is lower than that of the control group (77.4%), indicating slightly impaired developmental competence of microinjected embryos. The positive mutation rate of the microinjected embryos is 50.0% (8/16), much higher than the selection threshold (i.e. 10%). These results revealed that Set 1 is an effective ZFN set to induce mutations at the targeting site of rabbit ApoCIII. Therefore Set 1 was chosen for in vivo production of ApoCIII KO rabbits.
Production of ApoCIII KO rabbits 
The mRNA of ZFN Set 1 was microinjected to the cytoplasm of rabbit pronuclear stage embryos, and transferred 145 microinjected embryos to 7 pseudo-pregnant recipient rabbits (Figure 4A). Freshly flushed embryos (n=75) without ZFN microinjection were transferred to recipients (n=6) as the control group. After 30 days of gestation, 21 kits were born in the microinjected group. PCR sequencing identified five (apoC3-R1, R9, R11, R12 and R16) as positive KO kits (Figure 4B). The term rate calculated as total term kits/total embryos is 14.5% (21/145), whereas the rate is 29.3% (22/75) in the control group. The KO rate calculated as total KO kits/total term is 23.8% (5/21). One KO kit (apoC-R16) died five days postpartum (20.0%, 1/5). The indelsat the targeting site include two insertions and three deletions; they are&#916;1, +1, +1, &#916;20 and &#916;21, for Rabbits # R1, R9, R11, R12, and R16, respectively (Figure 4B).
Bioinformatics analysis was used to identify potential ZFN off-targets with 7 or fewer mismatches to Set 1 sequence, as described previously17. Only one predicted possible off-target site, located on Chromosome 14, was identified with 7 mismatched bps. PCR assay detected no off-target events in any of the founder animals.

Watch this Scientific Journal Video about Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases at JoVE.com

Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases

Recent development in gene targeting tools makes production of knockout (KO) rabbits possible. In the present work, we generated five Apolipoprotein (Apo) C-III KO rabbits using Zinc Finger Nucleases (ZFN). This work demonstrated that ZFN is a highly efficient method to produce KO rabbits.

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins ...

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18.0K Views.  University of Michigan Medical Center. Recent development in gene targeting tools makes production of knockout (KO) rabbits possible. In the present work, we generated five Apolipoprotein (Apo) C-III KO rabbits using Zinc Finger Nucleases (ZFN). This work demonstrated that ZFN is a highly efficient method to produce KO rabbits.

Video: Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Blind Endotracheal Intubation in Neonatal Rabbits

The newborn rabbit is a useful animal model for various pathologies and procedures. Airway management of the rabbit is complex due to its anatomical characteristics, which is further complicated in the case of the newborn. Of the different methods of advanced airway management, endotracheal intubation is less aggressive than tracheostomy, and is more feasible than supraglottic management given the lack of supraglottic devices of such a small size. As direct glottis visualization is very difficult in animals this size, this blind intubation model is presented as an effective alternative, especially for experiments requiring prolonged anesthesia. Using this method, we performed blind intubations with a 90% success rate.

We describe a technique of endotracheal intubation in newborn rabbits after esophageal catheterization with a gastric tube.

The newborn rabbit is a useful animal model for various pathologies and procedures. Airway management of the rabbit is complex due to its anatomical ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...

Three-Dimensional Finger Motion Tracking during Needling: A Solution for the Kinematic Analysis of Acupuncture Manipulation

Three-dimensional (3D) motion tracking has been used in many fields, such as the researches of sport and medical skills. This experiment aimed to use 3D motion tracking technology to measure the kinematic parameters of the joints of fingers during acupuncture manipulation (AM) and establish three technical indicators "amplitude, velocity and time". This method can reflect the operation characteristics of AM and provide quantitative parameters along three axes of multiple finger joints. The current evidence shows that the method has great potential for future applications such as the study of the dose-effect relationship of acupuncture, teaching, and learning of AM, and the measurement and preservation of famous acupuncturists' AM.

This experimental method describes a solution for the kinematic analysis of acupuncture manipulation with three-dimensional finger motion tracking technology.

Three-dimensional (3D) motion tracking has been used in many fields, such as the researches of sport and medical skills. This experiment aimed to use 3D ...

A Modified Technique for Arteriovenous Fistula Construction in Rabbits

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury to the veins and arteries during the operation and hemodynamic changes can lead to intimal hyperplasia, leading to juxta-anastomotic stenosis. To reduce injury to the veins and arteries during the operation, this study proposes a new modified no-touch technique (MNTT) for AVF construction that can decrease the rate of juxta-anastomotic stenosis and improve the AVF patency. To unravel the hemodynamic changes and mechanisms of the MNTT, this study presented an AVF procedure using this technique. Although this procedure is technically challenging, 94.4% procedural success was achieved after adequate training. Ultimately, 13 out of 34 rabbits had a functional AVF 4 weeks after the surgery, leading to a 38.2% AVF patency rate. However, at 4 weeks, the survival rate was 86.1%. Ultrasonography showed active blood flow through AVF anastomosis. Furthermore, the spiral laminar flow was observed in the vein and artery near the anastomosis, suggesting that this technique may improve the hemodynamics of the AVF. On histological observation, significant venous intimal hyperplasia was observed at the AVF anastomosis, whereas no significant intimal hyperplasia was observed at the proximal external jugular vein (EJV) of the anastomosis. This technique will improve the understanding of the mechanisms underlying the use of MNTT for AVF construction and provide technical support for the further optimization of the surgical approach in AVF construction.

The present protocol proposes the creation of an arteriovenous fistula in rabbits using a modified no-touch technique. The technique involves the side-to-side anastomosis of the common carotid artery and external jugular vein without the dissection of the perivenous tissues or cutting off the artery.

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury ...