200 micrograms of pure plasmid DNA (e.g. Qiagen kit-purified) at a concentration of 1 µg/µl or greater, or BAC DNA prepared by this protocol.
Information on the restriction enzymes which will allow us to cut and gel-purify a plasmid insert from the backbone.
A photo of a gel with markers showing the plasmid insert liberated from the plasmid backbone by restriction digest with the enzymes we will use to gel-purify the insert.
A demonstration that 1 copy or less of the transgene can be detected by Southern or PCR in a background of mouse genomic DNA. If PCR will be used, the assay must be performed on 1, 0.1, 0.01, 0.001 transgene copy equivalents to demonstrate the sensitivity of the assay and to reveal any background problems--a point of no detection should be reached in this series. Use this calculator to determine the amount of transgene per unit of genomic DNA.
What We Will Do
We will gel-purify plasmid inserts.
We will inject the insert or BAC into fertilized one-cell embryos.
While we don't provide a guarantee, our goal is to deliver at least 3 founders. We have an outstanding record of success.
We will house the mice until they are safe to transport.
What You Will Provide Part II
You will receive and house the mice.
You will perform your validated assay on the DNA from the mice we provide, and tell us which mice are transgenic. We recommend that you include control DNA samples, consisting of negative control (nontransgenic mouse genomic DNA) and the series of positive controls (nontransgenic mouse genomic DNAs spiked with 1, 0.1, 0.01, 0.001 copies per genome of your linearized transgene). If multiple primer pairs fail to detect founders by PCR, Southern blot analysis should be performed. Southerns are less susceptible to false negatives and worth the effort.
We ask that you acknowledge the Case Transgenic and Targeting Facility in seminars and publications.
Investigators need an IACUC protocol from their institution to receive mice from the core. CWRU IACUC protocols need to specify use of the Case Transgenic Core. Investigators do not need an IBC protocol of their own, normally: the review for recombinant DNA in animals will be conducted on the information submitted through online ordering for the transgenic core, and will be added to the core's IBC protocol as an amendment if approved. You may be contacted by the IBC for additional information as part of rDNA review after submission of the order. IBC approval is required prior to initiation of injections.
From the injection date of DNA, it will be a minimum of 6 weeks before we will be able to deliver mice to you (almost 3 weeks for gestation, and at least 3 weeks of postnatal growth before weaning).
Currently (2/26/17), the core is injecting constructs 4 weeks after reagents are ready to go. AFter ordering constructs are reviewed and approved by the IBC rDNA committee, DNA is received and fragments are gel-purified by the core, genotyping assays are reviewed. Once the reagents are ready to inject, mice to fulfill the service are ordered and received from Jax.
$3,000 for plasmid inserts <10kb into B6SJL. An additional $1,500 for BACs and plasmid inserts >10kb, and/or an additional $1,500 for strains other than B6SJL: for example, a small plasmid into B6SJL would be $3,000, a small plasmid insert into FVB would be $4,500, a BAC into C57BL/6J would be $6,000.
Local, non-CWRU orders will be charged an additional $200 for packing and transporting mice. Non-local orders will be charged $200 and the costs of transportation.
You will need the following information to order
PI name, address and contact info
IACUC protocol number
Insert size (kb)
Vector backbone size (kb)
Enzyme to free insert
Type of vector (plasmid, BAC, etc)
Mouse strain to inject
As well as (for rDNA/IBC regs)
Purpose and design of the animal experiments
Name, function and species of the gene
Name of the backbone vector
A weblink to the backbone vector map/sequence
Whether the gene is involved in infectious disease in normal healthy adult humans (yes/no)
Whether the gene presents any hazard to health or the environment (yes/no)
A .pdf file of a map of the construct
Success Rates in Generating Transgenic Mice
For both small (plasmid) and large (BAC) transgene constructs, the core generates an average of over 5 DNA-positive founder transgenic animals per construct.
Frequently Asked Questions About Transgenics
What is pronuclear injection?
Pronuclear injection of fertilized eggs
is the most common and convenient way to make transgenic mice. However,
it is important to be aware of some of the shortcomings of the technique.
This method results in multiple transgene copies inserted at a single
locus. Occasionally, late integration results in mosaic founders. Multiple
independent insertions in a founder occur with low frequency. About 1
in 10 insertions result in a phenotype due to disruption of the host genome.
In addition to insertional mutations, deletions and complex rearrangements
of the host genome can occur. Expression levels do not correlate well
with copy number. Not all integrated transgenes are expressed. In addition,
expression may be lost due to methylation arising during breeding of the
line. For analysis of transgene function, it is wise to work with three
independent transgenic lines because of effects of the host genome on
the transgene, and of the integration event on the host genome. Despite
these shortcomings, pronuclear injection is often the quickest way to
achieve your goals.
The classic transgene design includes an enhancer and promoter, the mRNA coding sequence and a complete set of poly-adenylation signals. A splice donor, intron and splice acceptor between the coding sequence and the poly-adenylation signals often increases expression. The construct should be designed so that the transgene insert can be purified away from plasmid backbone by gel purification. (The University of Virginia Health System Gene Targeting and Transgenic Facility has a diagram illustrating the principle.)
You might also consider a transgene based on large genomic fragments--BAC transgenes. A thorough discussion of the advantages and disadvantages of small, plasmid-based transgenes, BAC transgenes and gene targeting can be found here.
Which strain of mice do we inject?
For most transgenics, we recommend B6SJL hybrid mice. However, we can inject a variety of strains. We routinely inject the inbred strains FVB and C57BL/6J in addtion to B6SJL hybrids. If your experimental goals require the use of other strains of mice, please contact us. For special projects, we have made or are making transgenics in B6CBA and B6C3H hybrids, and in investigators' knockout strains.
Are there alternatives to pronuclear injection?
ES cells can be stably transfected by retroviruses,
random insertion, or by targeted insertion. Retroviruses modified for
ES cells are required for expression in ES cells. Multiple independent
single copy insertions can be obtained with retroviruses. Electroporation
of ES cells most often results in single copy insertion, and cell lines
that give appropriate expression can be selected in culture. Several independent
lines should be analyzed in mice if random insertion is used. Targeting
the transgene into a well-characterized locus (e.g. HPRT) is a viable
alternative. All commonly used ES cell lines are from the 129
strain of mice.
Will we inject your BAC?
Certainly. The protocol for BAC purification for injection is here. BAC transgenes have numerous advantages over plasmid transgenes in that they give more normal expression levels, are not susceptible to epigenetic inactivation, and give expression proportionate to copy number. Genomic BAC clones of mouse or human DNA often give normal spatial and temporal expression without the need to characterize promoters and enhancer combinations. BAC clones can be manipulated by recombineering to insert or delete sequences. BAC genomic clones can be used to complement mutations. Human and mouse (several strains) genomic BAC libraries have been end-sequenced and tiled on the genome. The BAC tiling of the different genomes can be seen at genome.ucsc.edu, and can be obtained often within 48 hours from bacpac.chori.org Except for exceptionally large genes, the size of the clones is sufficient to include the entire gene--additional genes can be deleted, if present. BACs are relatively stable in E. coli and relatively easy to purify. Examples of the use of BAC clones for Cre and GFP expression and genetic complementation can be seen in Scott et al., PNAS 102: 16472. If knockouts for a gene already exist, expression of mutant genes from a BAC can be an alternative strategy to construction of knockin mutants by gene targeting.
Why are my mice funny colors?
A number of coat color alleles are segregating
in the B6SJL F2 hybrid mice. F1 hybrids of the SJL
inbred strains were mated to produce F2 zygotes that were injected with
DNA. SJL is A/A, rd/rd, p/p, and c/c. C57BL/6J is a/a and wild type at
the other loci. A
(agouti, dominant brown coat color), is the wild-type version of a
(non-agouti, recessive black coat color). rd
(recessive retinal degeneration) is a mutation in a phosphodiesterase
expressed in rod cells. p
(recessive pink-eyed dilution) decreases pigmentation. c
(recessive albino) causes loss of pigmentation. p/p in the presence of
A results in yellow mice. p/p in the presence of a/a leads to gray mice.
Thus, a rainbow of white, brown, black, gray and yellow mice are found
in the F2 C57/SJL mice. FVB is c/c and rd/rd: albino and blind.
Blindness, Deafness and Glucose Intolerance in Transgenic Mice The mutant rd1 allele of Pde6b is common in inbred strains, and segregates in the B6SJL transgenic mice mice we generate, giving rise to both sighted and blind transgenic animals. Some inbred strains have hearing deficits. The age-related hearing loss mutation of Cdh23 (Cdh23ahl) mutation is present in the C57BL/6J background and thus will be present in all transgenic mice we make on this background, and will segregate in the B6SJL transgenics we generate, giving rise to transgenics with hearing loss and others which are normal. Many inbred strains have the age-related hearing loss mutation (Cdh23ahl), which causes degeneration of hearing starting at about 10 months of age, depending on the genetic background (Johnson et al., (2000) Genomics 70:171 (.pdf)). The C57BL/6J strain is mutant for nicotinamide nucleotide transhydrogenase, which contributes to glucose intolerance. This trait will be present in transgenics made on C57Bl/6J and will segregate in transgenics made on B6SJL.
Alternatives For High Throughput
Our regular service for transgenics is structured to provide certainty that the client will obtain sufficient founders for every construct. We also offer an alternative service for batches of 4 or more constructs which is structured on payment for each day of microinjection (per-day injection agreement). In this batch service, the client assumes the risk that no founders may be produced on a day of microinjection. This service is limited to batches of 4 or more constructs, the inserts must be less than 10kb in size, the client must purify the DNA inserts by our method, the strain of mice must be C57Bl/6JxSJL F2, only one construct will be injected on a given day, and the client assumes the risk for nonproductive injection days. The cost is $1750 per injection day with a minimum $200 animal transport charge for non-CWRU locations. Although we will take batches as small as 4 constructs, this option works best for the client with larger numbers of transgenes.
Our protocols and worksheets for insert isolation and purifcation for the per-day injection agreement are here.