| What types of genetic monitoring testing does Charles
River Genetic Testing Services offer? |
Charles River Genetic
Testing Services provides DNA-based testing to facilitate the periodic
genetic monitoring of transgenic and knockout strains of laboratory animals.
Charles River Genetic Testing Services employs the techniques of DNA
amplification using both PCR (polymerase chain reaction) and real-time QPCR
(quantitative PCR). In addition, Charles River Genetic Testing Services offers
DNA profile testing using microsatellites for strain characterization and
differentiation, maintenance of strain purity, and
speed congenics (MAX-BAXSM). Our services include genotyping for
a specific gene by PCR, SNP analysis, zygosity and expression testing using
QPCR, and genome scans by microsatellite analysis.
-
MAX-BAXSM (Marker Assisted Accelerated Backcrossing) is used for
the application of speed congenics. Candidate breeders are genotyped at each
generation, and those with the highest percentage of the desired strain are
selected to father the next generation. This can greatly reduce the number of
backcross generations needed to produce a research model, cutting both time and
cost significantly.
-
Zygosity Testing, utilizing QPCR, can discriminate the two-fold difference
in gene copy number. This is a quantitative assay for the determination of
transgene zygosity.
-
Expression Testing, utilizing RT-QPCR (reverse transcriptase QPCR), can
measure the mRNA accumulation and therefore determine the in vivo
transcriptional activity of a transgene.
- NOD (non-obese diabetic disease model) mice exhibit disease due to a
polygenic mechanism involving numerous recessive genes. Genetic Testing
Services can determine the existence of this polygenic mechanism through PCR
analysis of 13 linkage markers.
- SCID (severe combined immunodeficiency) is an autosomal recessive mutation
that can spontaneously occur. Genetic Testing Services can determine the
existence of this point mutation utilizing PCR with an additional digestion
step.
|
| Can I have genetic testing performed while my animals
are being raised at Charles River? |
| Yes, Charles River Laboratories Transgenic
Services and Genetic
Testing Services can provide full breeding and genotyping services, as well
as develop new tests should existing tests fail or be sub-optimal. Samples
taken from animals raised at your facility can also be sent to Charles River
Genetic Testing Services directly for genetic monitoring. |
| What type of samples should I send? |
| Charles River Genetic Testing Services requests tail biopsies between
0.5 - 1.0 cm in length, immersed promptly in 70% ethanol and refrigerated
prior to shipping. This is our preferred test article, as it generally provides
sufficient high-quality DNA. We can also accept toes or ear clips; but the
amount of tissue from a standard earpunch is not sufficient. All samples should
be placed on ice packs and shipped overnight. Please do not process or freeze,
as this increases the risk of DNA degradation. However, if only frozen samples
are available, these may be shipped overnight, packed with dry ice. Please
contact Genetic
Testing Services (518-286-0016) prior to shipping samples.
Expression Testing may require other types of tissue samples, as determined
by the investigator. Again, please contact Genetic Testing Services
(518-286-0016) regarding sample type and collection conditions prior to
shipping samples. |
| How do I send samples? |
| Tail biopsies (0.5-1.0 cm in length) should be immersed in 70% ethanol in
microcentrifuge tubes and shipped overnight packed with cold packs. The first
page of the sample submission form should be included with the shipment. Please
call Charles River Genetic
Testing Services (518-286-0016) prior to shipping samples so we know when
we can expect to receive them. This also allows us to take prompt action if
your shipment does not arrive when expected. |
| What is the lead-time for receiving services? |
| Charles River Genetic Testing Services accepts delivery of samples
Monday through Friday. As samples cannot be received on Saturday or Sunday,
please take this into account when planning your shipping schedule. Following
receipt, Charles River Genetic Testing Services will begin processing samples
promptly. Charles River understands the need for timeliness of sample
processing in order to facilitate your work. |
| What is the turnaround time for Genetic Testing
Services? |
|
PCR test results have an approximate 5-10 business day turnaround time
following receipt of samples, provided the protocol has already been
established at Charles River Genetic Testing Services and is working
effectively. (Please note that Transfer and Validation of new protocols may
require an additional 5-14 days.). Results from
DNA profile/characterization testing, a technique generally used for
characterizing background/inbred strains and for MAX-BAXSM/speed
congenics, is reported in 2-3 weeks. Results from
Zygosity and Expression Testing production samples are typically reported
approximately 2 weeks following development of the assay. Development of new
assays may require an additional 2 weeks. |
| How do I get my results? |
| Results are generally emailed, although they can also be mailed or faxed
to the customer directly. ILIMS, a system allowing clients to access their
results directly over the Web, is also an option for accessing results. Some
results are verbally discussed initially, with a final report sent by
e-mail. |
| Can Charles River Genetic Testing Services
use my protocol? |
| Charles River Genetic Testing Services can use your protocol, optimize an
existing protocol, or develop a protocol for the genetic monitoring of your
production samples. Genetic Testing Services does require that you Transfer and
Validate an existing protocol with Charles River Genetic Testing Services
first. A Transfer and Validation is an initial study that validates the
efficacy and utility of an assay and is performed prior to accepting production
samples. In order to guarantee successful transfer and validation of an assay,
we suggest that all requested information and materials be submitted at least
one month prior to the receipt of production samples. There is a one-time cost
associated with the Transfer and Validation of an existing protocol. Transfer
and Validation forms can be downloaded from this website for your use. Please
contact Genetic Testing Services for more information regarding pricing and
submission of these and other forms at (518) 286-0016. |
| What if I don't have a protocol or my protocol doesn't
work? |
Charles River Genetic Testing Services has the expertise to transfer
and/or optimize an existing protocol, or develop a new protocol to suite your
genetic monitoring needs.
Please contact Genetic
Testing Services at (518) 286-0016 for more information. |
| What if I don’t have a protocol and don’t have time for
the Transfer and Validation? |
Many vectors designed for transgenics use the same, well- characterized
elements: selectable markers, reporter genes and Cre recombinase. Charles River
Genetic Testing Services has developed and optimized both PCR and QPCR assays
for the most common elements (listed below). Investigators can assay the
selectable marker, reporter gene or recombinase without incurring transfer and
validation or assay development fees.
- Neomycin
- GFP
- LacZ
- Cre
- Luciferase
This approach can be used in situations where there may have been a mix-up
between knockout and control animals. |
| Will Charles River provide me with a copy of the
protocol? |
| The assays we optimize remain the property of the client and are not
provided to anyone else. If we perform a transfer and validation for you, we
will be happy to provide you with a copy of the optimized assay. We retain a
copy at Charles River Genetic Testing Services and reserve the right to use it
for other clients. Clients who are using an existing protocol developed for
another client will not be given a copy of the protocol. |
| Should I test males or females for
MAX-BAXSM? |
| In general, we prefer to test males because they can be harem-mated,
allowing for more rapid generation of sufficient numbers of offspring for the
next-generation testing. However, it is generally necessary to backcross one
round of females in order to fix the X-chromosome. However, if the gene of
interest is X-linked, all the animals being assayed for backcrossing must be
female. For more information, please read the following document entitled Guidelines
for Breeding Animals for MAX-BAX. |
| How many animals should I test for
MAX-BAXSM ? |
| In each generation, the percent of recipient strain in the offspring will
vary around the average as a bell-shaped curve. It is necessary to provide a
sufficiently large sample so that the animals with high levels of recipient DNA
have a good chance of being detected. Statistical simulations show 16 animals
to be an effective number. In practice, good results can be obtained with about
10 animals per generation. For more information, please read the following
document entitled Guidelines
for Breeding Animals for MAX-BAX. |
| When should I start testing animals for
MAX-BAXSM ? |
If the initial animals are already on a defined background, the F1 progeny
(of the founder animal and a mate of the recipient strain) will all be 50%
recipient strain, so there is no advantage in performing a microsatellite
analysis at this point. If the founder animal is homozygous for the gene of
interest, the progeny are obligate heterozygotes, and genotyping for the gene
of interest is also not necessary for this generation. If the initial animals
are on a hybrid background and the desired recipient strain is one of the
strains contributing to this background, MAX-BAXSM genotyping of the
F1 generation can identify animals with a higher-than-average percentage of the
recipient strain. For more information, please read the following document
entitled Guidelines
for Breeding Animals for MAX-BAX.
|
| Should I use RFLP or microsatellites for strain
characterization? |
| For mouse strains, the microsatellite panels are superior to RFLP analysis.
We assay up to 110 microsatellites, versus 20-40 RFLP polymorphisms. In
addition, the chromosomal location of each microsatellite marker is known, and
the assay can determine whether the animal in question is heterozygous or
homozygous for each marker. |
| How should I decide between a PCR or QPCR genotyping
assay? |
|
PCR assays can determine zygosity of knockouts, knock-ins, and point
mutations (with subsequent restriction digest). They can also determine
carrier status (but not zygosity) of transgenes.
QPCR assays can determine zygosity (hemizygote versus homozygote) of a
transgene. The assay can also be used to determine trangene copy
number.
|
| Can I get the Charles
River microsatellite primer sets? |
| At Charles River Genetic Testing Services, we have tested a large number of
microsatellite markers and established panels of markers which differentiate
between certain strains. This required extensive research and development.
Therefore, we do not give out our markers, but do offer marker analysis as a
service. |
