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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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. 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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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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.

<table><tbody><tr><td>APOC3-ZFN</td><td>Sigma Aldrich</td><td>CSTZFNY-1KT</td><td>Target Gene: APOC3, Lot Number: 12201118MN</td></tr><tr><td>mMESSAGE kit</td><td>Invitrogen</td><td>AM1344M</td><td>mRNA synthesis</td></tr><tr><td>MEGAclear Kit&amp;nbsp;</td><td>Invitrogen</td><td>AM1908M</td><td>mRNA purification</td></tr><tr><td>Follicle-stimulating hormone</td><td>Bioniche Life Sciences</td><td>Folltropin-V</td><td>Treating embryo donor rabbits</td></tr><tr><td>Human chorionic gonadotropin</td><td>Intervet</td><td>Chorulon</td><td>Treating embryo donor rabbits</td></tr><tr><td>Gonadotropin-releasing hormone</td><td>Prospecbio</td><td>HOR-255</td><td>Treating embryo recipient rabbits</td></tr><tr><td>Earle&#039;s Balanced Salt Solution (EBSS)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>SH30029.02</td><td>Embryo culture, base medium</td></tr><tr><td>MEM (nonessential amino acid)</td><td>Sigma Aldrich</td><td>M7145</td><td>Embryo culture, supplements</td></tr><tr><td>BME AMINO ACIDS solution</td><td>Sigma Aldrich</td><td>B6766</td><td>Embryo culture, supplements</td></tr><tr><td>Glutamine</td><td>Gibco</td><td>25030-149</td><td>Embryo culture, supplements</td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Gibco</td><td>11360-070</td><td>Embryo culture, supplements</td></tr><tr><td>Fetal bovine serum</td><td>Sigma Aldrich</td><td>12003C</td><td>Embryo culture, supplements</td></tr><tr><td>HEPES buffered TCM 199&amp;nbsp;</td><td>Gibco</td><td>12350039</td><td>Embryo manipulation medium</td></tr><tr><td>Incubator</td><td>Eppendorf</td><td>Galaxy 170</td><td>Embryo culture equipment</td></tr><tr><td>Micromanipulator&amp;nbsp;</td><td>Eppendorf</td><td>TransferMan NK 2</td><td>Embryo manipulation equipment</td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments Inc.</td><td>P-1000</td><td>Embryo manipulation equipment</td></tr><tr><td>Microinjector&amp;nbsp;</td><td>Tritech Research</td><td>MINJ-D</td><td>Embryo manipulation equipment</td></tr><tr><td>Borosilicate glass capillary tubes&amp;nbsp;</td><td>World Precision Instruments, Inc.</td><td>TW100F-6</td><td>Embryo manipulation supply</td></tr><tr><td>Euthasol (pentobarbitol sodium)</td><td>Virbac AH, Inc.</td><td>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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Universidad de Cartagena UDEC

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Medicine

18.1K 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

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Genetics

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Culturing and Manipulation of O9-1 Neural Crest Cells

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.

O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing.

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

Developmental Biology

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Biology

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily relative to traditional homologous recombination. The CRISPR-Cas9 system is particularly attractive when a single-point mutation is desired. The gap junction protein, Connexin 43 (Cx43), is encoded by gene Gja1, which has a single coding exon and cannot be spliced. However, Gja1 produces not only full-length Cx43 protein but up to six N-terminus truncated isoforms by a process known as internal translation, the result of ribosomal translation initiation at internal AUG (Methionine) start sites. GJA1-20k is the most commonly generated truncated isoform of Cx43 initiated at the AUG codon at position 213 of Gja1 mRNA. Because residue 213 occurs at the end of the last transmembrane domain of Cx43, GJA1-20k is effectively the 20 kDa C-terminus tail of Cx43 as an independent protein. Previous investigators identified, in cells, that a critical role of GJA1-20k is to facilitate trafficking of full-length Cx43 gap junction hemichannels to the plasma membrane. To examine this phenomenon in vivo, a mutant mouse with a Gja1 point-mutation was generated that replaces the ATG (Methionine) at residue 213 with TTA (Leucine, M213L mutation). The result of M213L is that Gja1 mRNA and full-length Cx43 are still generated, yet the translation of Gja1-20k is significantly reduced. This report focuses on choosing the restriction enzyme site to develop a one amino acid mutated (Gja1M213L/M213L) mouse model. This protocol describes genetically modified mice by the CRISPR-Cas9 system and rapid genotyping by combining PCR and restriction enzyme treatments.

The present protocol describes a single M213L mutation in Gja1 that retains full-length Connexin43 generation but prevents translation of the smaller GJA1-20k internally translated isoform.

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily ...

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

Summary

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Chapters in this video

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Culturing and Manipulation of O9-1 Neural Crest Cells

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

TGF-β-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein