FAQ's
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.

Transgene Design
     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.)

Transgene Detection
     You will need a strategy to detect the transgene in mice, in order to identify founder mice and the transmission of the transgene.

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 and C57BL/6J 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 1 (Ahl1) 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 1 (Ahl1) mutation, 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.

• Investigators need an IBC protocol from CWRU
• Investigators need an IACUC protocol from their institution.

• An account number.
• 200 micrograms of pure plasmid DNA (e.g. Qiagen kit-purified) 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 you will be able to detect transgenic founders: Either a Southern blot of wild type mouse genomic DNA, and wild type mouse genomic DNA spiked with an appropriate amount of transgene, probed with the transgene. OR A photograph of a gel showing PCR of wild type mouse genomic DNA, and wild type mouse genomic DNA spiked with an appropriate amount of transgene.

• 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 a proven record of success. We have an outstanding record of success.
• We will house the mice until a few days after birth.

• You will house the weaned 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 two positive controls (nontransgenic mouse genomic DNAs spiked with one and ten 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.

• $2,500 for plasmid inserts <10kb into B6SJL. An additional $1,250 for BACs and plasmid inserts >10kb, and/or an additional $1,250 for strains other than B6SJL: for example, a small plasmid into B6SJL would be $2,500, a small plasmid insert into B6CBA would be $3750, a BAC into C57BL/6J would be $5000. An additional minimum of $200 for animal transport will be charged for non-CWRU orders.

• We ask that you acknowledge contributions of the Case Transgenic and Targeting Facility in seminars and publications.