Products and Services | Services for Genome Editing Knockout & Knockin Rabbit and Guinea Pig Model

 

Renova Life Inc. (RLI) provides the genome editing knockout and knockin rabbit and guinea pig service worldwide.

Rabbits are phylogenetically closer to humans, comparing to mice. Because of the anatomical, physiological, genetic and biochemical similarities between the rabbit and the human, this species is preferentially used in pulmonary, cardiovascular and metabolic studies, including airway obstructive disease, embolic stroke, arteriosclerosis, cholera, cystic fibrosis, neoplasia, diabetes, acute respiratory distress syndrome, malignant lymphoma, acquired immunodeficiency syndrome and hypercalcemia of malignancy.  As a classical experimental animal species, rabbit has several other advantages over some other animal species (e.g., monkey, pigs). It has a short gestation period (30-31 days), large litter size (4-12/litter) and can be housed conveniently in an indoor facility.   In addition to the use as an animal model, rabbit is also used for drug screening, antibody production, and the production of therapeutic proteins (bioreactors). Some proteins can be properly synthesized in rabbit milk, but not in other animal systems (e.g. cattle or goats) due to the post translation processing.

 

The guinea pig (Cavia porcellus) is a species of rodent belonging to the family Caviidae and the genus Cavia. They are used in research, primarily as models for human medical conditions such as juvenile diabetes, tuberculosis, scurvy, and pregnancy complications. Guinea pigs were popular laboratory animals until the later 20th century; about 2.5 million guinea pigs were used annually in the U.S. for research in the 1960s. In the past, they were widely used to standardize vaccines and antiviral agents; they were also often employed in studies on the production of antibodies in response to extreme allergic reactions, or anaphylaxis. Less common uses included research in pharmacology and irradiation. In 2004, the U.S.'s National Human Genome Research Institute announced plans to sequence the genome of the domestic guinea pig. The guinea pig was most extensively implemented in research and diagnosis of infectious diseases. Common uses included identification of brucellosis, Chagas disease, cholera, diphtheria, foot-and-mouth disease, glanders, Q fever, Rocky Mountain spotted fever, and various strains of typhus. They are still frequently used to diagnose tuberculosis, since they are easily infected by human tuberculosis bacteria. Because guinea pigs are one of the few animals which, like humans and other primates, cannot synthesize vitamin C, but must obtain it from their diet, they are ideal for researching scurvy. The guinea pig model proved a crucial part of vitamin C research. Complement, an important component for serology, was first isolated from the blood of the guinea pig. Guinea pigs have an unusual insulin mutation, and are a suitable species for the generation of anti-insulin antibodies. Present at a level 10 times that found in other mammals, the insulin in guinea pigs may be important in growth regulation, a role usually played by growth hormone. Additionally, guinea pigs have been identified as model organisms for the study of juvenile diabetes and, because of the frequency of pregnancy toxemia, of pre-eclampsia in human females.

 

We can quickly knockout or knockin a gene in the rabbits and guinea pigs, with a consideration of nuclease-mediated genome editing technologies as an alternative, since the homologous recombination-based knockout approach is not available due to lack of authentic embryonic stem cells in these two species. Several types of artificially constructed nucleases (e.g., TALEN, Cas9 and ZFN) can be engineered to recognize and cleave arbitrary sequences. When such nucleases (or their DNA or mRNA precursors) designed to target a specific site in the rabbit/guinea pig genome are microinjected into fertilized eggs, cleavage at the target site followed by imperfect repair can result in small deletions (and insertions, more rarely) of one or more base pairs. If the cut site is in the coding region of a gene, this will result in frameshift mutations downstream of the site, generating a knockout. The specific point mutations in the repair template can be introduced at the nuclease cleavage site, when a repair template with homology to the target site is present during the repair process, thus generating a knockin. Genome editing using TALEN or CRISPR/Cas9 can generate a knockout or knockin in as little as ~3 months in rabbits, and 5-6 months in guinea pigs.

 

Flow Chart of Gene Targeting Services in Rabbit and Guinea Pig


Service Description:

Animals
New Zealand white rabbit and Hartley short hair guinea pig will be used for generating nuclease-mediated knockout or knockin animals by TALEN and CRISPR-Cas9 technology in both species.

 

Strategy and vector design
We will design a nuclease-mediated strategy for the clients by providing which the gene is like to knockout or with which point mutation is desired to knockin in targeted gene. This includes identification of the type of nuclease (TALEN or CRISPR/Cas9) most suitable for your goals.  If CRISPR-Cas9 technology is applied, we will design the optimal targeted sites in the gene for a higher efficiency, but minimize off-target activity by trunked guide RNA via CRISPR/Cas9 technology. If the client has experience on construct designing, we will be more than happy to discuss a better solution and discount for knockout and knockin in rabbit and guinea pig model. For each targeted gene, we will design vectors against two or three target sites in the gene to ensure success. Genotyping assays based on PCR and sequencing will also be designed for the screening of knockout or knockin founder rabbits and guinea pigs.

 

Microinjection of TALEN and CRISPR-Cas9 mRNA into rabbit/guinea pig eggs
DNA vectors that express the desired nucleases will be constructed. To assure the success of knockout and knockin in these two species, we will perform prior experiments before the real experiment. We will test the efficiency of vectors in rabbit and guinea pig cell line and select the best one for embryo work. When optimal vector is selected in cell lines, RNA microinjection will be performed in fertilized eggs and injected embryos will be cultured in vitrointo blastocyst stage for further genome screening. This experiment can be assured that the constructs will work for generating KO and KI rabbits and guinea pigs after embryo transfer.

 

RNA: Nuclease mRNA will be transcribed in vitro. Relative capping and polyadenylation modification to mRNA will be completed to ensure its proper translation into protein in mammalian cells and embryos.

 

Pronuclear and or cytoplasmic injection to obtain founders: According experimental design, nuclease mRNA and/or donor DNA will be injected into fertilized eggs, followed by transferring injected eggs into the synchronized recipient to obtain F0 offspring. We guarantee a minimum of 3 knockout or knockin founders.  According to our experience, more positive founders will be generated by CRISPR-Cas9 gene editing technology.  We may request the bonus if client needs more founders after completion of the project. Usually, we will inject 150-200 eggs per optimal target site (selected from cells and cultured embryos) to achieve our target, in any case, we will target two or more sites with microinjection of 400-500 eggs per gene to reach our promise and guarantee.

 

If client prepares nuclease expression vectors (or mRNA products), it is recommended that 2-3 sites per gene is targeted in order to increase the chance of success. The verification of efficiency in cell lines are required.

 

Determining Knockout/Knockin founders
PCR and further sequencing will be performed to screen out knockout or knockin founder animals. For more convincing of screening results, we prefer client to identify the positive founders with the tissues collected from newborns, although we can provide the service for knockout and knockin screening upon the approval from the client. Animals carrying frame shift deletions/insertions on at least one allele are considered knockout founders. In some cases, an animal may be found to have both alleles of the target site mutated, but the chances for survival will be decreased in those double knockout animals.

 

Breeding service of founders
Breeding service will be a separate project to knockout/knockin project in rabbit and guinea pig model. Since the breeding project is longer than 6-8 months and the risk of uncontrollable factors is high during growth period of founder animals and breeding, we will discuss with the clients in details for a better breeding strategy.  Usually, we will raise the founder animals to sex maturity (rabbit female, 5-6 months, rabbit males, 9-10 months; guinea pig male and female, about 3 months), and then we breed the founders to wild type animals, and screen the offspring to obtain positive F1 offspring.
  
Service guarantee
When the nuclease expression vectors are designed and constructed by RLI, we will fully refund service charge if founder(s) fails to be generated. When the vectors are provided by the client, we cannot guarantee the generation of knockout or knockin animals, but when client provides the convincing data in cultured cells, and further confirmed by our embryo microinjection study, we will discuss with client for the guarantee in a scientific and professional manner. We will discuss the risk of knockout important gene on the X chromosome or any critical genes affecting or detrimental to fetal development.

 

Service Charge and Turnaround Time

Project overview with a full service


Milestones

Service

Price

Turnaround time

1

Strategy and vector design

Free

Two weeks

2

Nuclease expression vector construction for knockout

$3,000

5-6 weeks

Nuclease expression vector construction for knockin

$3,500

5-6 weeks

3

mRNA preparation and vitrification

$2,000

2-3 weeks

4

Generating  knockout founders  by TALEN or CRISPR/Cas9 micro-injection into fertilized eggs and ET

Rabbit

$75,000

2-3 months

Guinea pig

$70,000

4-6 months

Generating knockin founders by TALEN or CRISPR/Cas9 micro-injection into fertilized eggs and ET

Rabbit

$95,000

2-3 months

Guinea pig

$85,000

4-6 months

5

Identifying knockout founders

$3,500

3-5 weeks

Identifying knockin founders

$4,500

4-6 weeks

6

Breeding founders to obtain F1

Consulting RLI

6-8 months

 

Price Discount
For the second project from old client, or a new project introduced by old client, the service charge will be provided with a discount of 15% off. We encourage the clients to promote our services and reputation.

 

Shipping
Animals and related products are shipped from our China CRO animal facility in Jiangsu Province, China (about 100 miles north of Shanghai).The client shall be responsible for transportation of transgenic animals from RLI China subsidiary facility to designated location by clients, including the charges derived from quarantining, health paper, transportation and related costs. RLI will provide all assistance and efforts to complete the delivery. RLI will use professional animal transportation company “World Courier” to ship live animals worldwide.

 

IPs and Confidentiality
Client has all rights to own animals and related products generated from the service. Any service information is highly confidential without the permission from client.

 

If client provides own vectors, the intellectual property (IP) is absolutely belong to the client. RLI will not look into any of IPs without consulting and approval from the client.

 

Inquiries and Quote Requests

Please email info@renovalife.com  or fax to 301-576-5078 to inquire about our transgenic or gene editing services in rabbits and guinea pigs.

 

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Products and Services | Services for Genome Editing Knockout & Knockin Rabbit and Guinea Pig Model