For more information about or to request custom services, please call or email:

Ihab Abd-Elrahman, Ph.D

ihabae@savion.huji.ac.il

0548820831

We look forward to discussing your particular needs.

Spontaneous mutations constantly arise in every mouse colony. Some of these mutations may spread throughout the colony until all mice are homozygous for them. These mutations may affect the phenotype of the line. However, cryopreservation can greatly reduce the genetic drift’s impact.
 

The cryopreservation service preserves mouse lines by sperm freezing.

a)    Sperm will be produced from client’s activated  male and they will be frozen according to the well-established protocol.

b)    At least 4 males of breeding age (3-8 months) of proven fertility are required to preserve a line.

c)    At least 20 straws of sperm from three male mice are stored in two separate locations.

d)    Quality control of frozen sperm is assessed by thawing one straw of frozen sperm for IVF, with wild-type oocytes. The resulting embryos are transferred to pseudo pregnant females to ensure they produce viable offspring.

e)    Embryo tissue samples are provided to the client for PCR verification of the strain.

Cryopreservation

Cryopreservation of mouse strains serves a number of important functions:

•It provides reduction in number of mouse cages and reduction of associated maintenance costs.

•For strains that are difficult to replace, it provides insurance against disaster or pathogen outbreak.

•Cryopreservation provides insurance against genetic drift.

Genetic drift is not trivial. Spontaneous mutations constantly arise in every mouse colony. Some of these mutations may spread throughout the colony until all mice are homozygous for them. These mutations may affect the phenotype of the line. However, cryopreservation can greatly reduce genetic drift’s impact.

We strongly recommend that you cryopreserve any mouse strain that is critical to your research that either you created yourself, or that may be difficult to reacquire. If a breeding error, genetic drift, loss of phenotype, or a pathogen outbreak hits your colony, and you have the strain cryopreserved, you can recover the original strain and continue your studies with minimal loss of time.

The cryopreservation service preserves mouse lines by sperm freezing. Keep in mind that we use wild-type oocytes to thaw the cryopreserved sperm, and the result will always be heterozygote mice.

At least 20 straws of sperm from four male mice are stored in two separate locations (note: males are sacrificed for sperm collection). Quality control of frozen sperm is assessed by thawing one straw of frozen sperm for IVF, with wild-type oocytes. The resulting embryos are transferred to pseudo-pregnant females to ensure they produce viable offspring, at least till E14.5. Embryo tissue samples are provided to the researcher for PCR verification of the strain.

Procedure

In order to preserve a line, the following is required:

• Complete the Cryopreservation request form - frozen/thawed mouse sperm.

•Please be aware that the strain name entered on the request form will be the name used on the straw labels (maximum of 16 characters).

• Complete one request form for each strain required to be frozen.

•At least 4 males of breeding age (3-8 months) of proven fertility are required. For any mouse older than that, we cannot assure sperm quality and we will only perform the task after specific request from the P.I. and under their responsibility. 

•A time table will be given.  The researcher is responsible to hold each male in a separate cage with one female for 5 days for the renewal of the sperm cells, separate the males from the females and keep each mouse individually for 5 more days before sending the mice to the GEMM unit.

•When transferring the cages of male mice for freezing, the order number (which you will receive by e-mail) must be clearly written on the cage label.

In the case of complicated genotypes (more than one mutation), or if there is strain information that we should be aware of, please contact the unit management before initiating the order.

In the event of a waiting list for freezing, we will notify you, and we appreciate your patience and cooperation.

MOUSE DNA TRANSGENES INJECTIONS

a)    The quality of the DNA preparation used in microinjection has a direct effect on the efficiency of DNA integration.

Each of the following parameters will affect the efficiency of DNA integration:

i) The injected fragment must be free of flanking plasmid sequences (which may be toxic).  Therefore, the construct should be designed so that the insert for microinjection can be excised without vector sequences.

ii) The DNA must be intact, without accompanying degradation, in order to prevent integration of partial constructs.

iii) The DNA must be free of contaminants such as phenol, ethanol, enzymes etc., which may harm the embryo.

iv) Solutions used in DNA purification and dilution buffers should utilize fresh, high quality sterile reagents, and Water for Embryo Transfer (Sigma W1503) must be used.

v) It is essential that the DNA solutions not contain any particulate material that may clog the microinjection needles.

vi) DNA concentration must be measured precisely in order for the final concentration for injection (2 ng/ul) to be accurately determined. The final dilution of DNA in injection buffer will be performed by the transgenic facility personnel.

vii) 50 ul purified DNA at a high concentration (50-100 ug/ml) should be delivered to the transgenic lab ten days before the assigned microinjection session, to check for DNA purity.

viii) It is highly recommended that you prove that your construct is expressed in vitro by an appropriate cell line. If this has not been shown for your construct, state it in your application.

b)    DNA will be injected into one of the pronuclei of the zygote of C57Bl6 background mouse and transferred to the pseudo pregnant female mouse.

c)    After 3 weeks weaning, pups will be delivered to the client for genotyping

a) A single guide RNA (sgRNA) recognizes a genomic region followed by 5’-NGG-3’ PAM sequence, which recruits the Cas9 DNA endonuclease. This introduces a double-stranded break that is repaired by:

i) Non-Homologous End Joining (NHEJ), an error prone pathway that can result in the creation of Indels that can disrupt the gene.

ii) Or by homology directed repair (HDR) in the presence of a donor construct.

b) Clients will provide us with specific Guide RNA that  we anneal with constant Tracer RNA and then mixed with Cas9 enzyme that we provide.

c) Guide RNA and Cas9 enzyme is introduced directly to the zygote by electroporation.

d) Electroporated zygote is transferred to the Embryo Recipient Pseudo-pregnant mouse.

e) After 3 weeks, weaned pups will be delivered to the client for genotyping.

f) Conditional Alleles:

i) Insertion of LoxP sites to flank an exon (exons) of interest is possible using CRISPR technology, but can be problematic due to low event probability.

ii) The most common method involves electroporation of two separate sgRNAs, targeted to sequences flanking an exon of interest and two ssODN donors, each containing a LoxP site flanked by short (40–80 bases) homology arms.

iii) Both LoxP sites must be inserted into the same allele. Efficiency tends to be low, and a variety of mutations (including the full unconditional knockout, single LoxP insertions, double LoxP insertions located in trans, deletions resulting from non-homologous end-joining –NHEJ- and insertion of mutated LoxP sites) are commonly detected.

iv) Alternative strategies using the Strandase enzyme kit could be used.

v) Strandase enzyme can digest a sence or antisence DNA strand that is produced by simple PCR, performed with phosphorylated primer.

vi) This technology allows us to perform Homology Directed Repair (HDR) with up to 5 kb donors, resulting in reduced tendency to randomly integrate into the genome and improved gene editing efficiency.

g) Off-targets:

i) CRISPR mice have been reported to have far fewer off-targets then cell lines

h) Guide RNAs, Tracer RNAs, Cas9 enzyme and HDR oligos:

i) We work with molecules produced by IDT.

ii) Guide RNAs and HDR oligos are provided by client, whereas Tracer RNAs and Cas9 enzyme are provided by us.

i) Testing new Guide RNAs and HDR donors:

i) Before starting the project proper, we can electroporate a number of embryos (about 50), and leave them to develop in vitro to blastocyst stage.

ii) The embryos can then be analyzed for the presence of the mutation, and for indels at the cut sites.

iii) If indels persist in a satisfying quantity (percentage) we will complete the project and transfer the electroporated embryos.

j) GEMM facility guarantees.