Microinjection Services
The Microinjection services offered by LASEC in CUHK are comprised of technical expertise, centralised facilities, sophisticated instruments, which is necessary to perform genetic modification and to produce genetically engineered mouse models (GEMMs).
Typically, a DNA construct or CRISPR-Cas9 reagents submitted by USERS are microinjected into the pronucleus and/or cytoplasm of mouse zygote of desired inbred or specialty strains. After brief culture, all the surviving embryos are transferred to the oviducts of pseudopregnant female mice and carried to term. The average gestation time for mice is 19 to 21 days. Once the pups are born and weaned, tailed, and numbered by LASEC personnel at 3-4 weeks of age. The researcher will be required to perform DNA isolation and analyse the genome editing by polymerase chain reaction (PCR), sequencing or Southern blotting to identify transgenic founder mice carrying the intended mutation.
Microinjection service is an essential step in the process to achieve the following complex genetic modifications and mouse model creation:
Random DNA transgenesis
Foreign DNA is injected into fertilized mouse oocytes, which are borne by surrogate mothers. Part of the new-borns will have integrated the foreign DNA into their own genomes.
Endonuclease-mediated site-specific genome editing
> Incorporation of point mutation (SNP)
> Insertion of a short fusion tag, such as HA or Flag
> Introduction of stop codon
> Creation of precise DNA deletion
> Insertion of loxP sites around one or more exons
> Swapping of the murine gene with its human ortholog
> Insertion of exogenous coding sequences encoding fluorescent proteins or Cre
Double-stranded plasmid donor DNA (Generally > 5 kb)
> Insertion of large fragment exceeding 5 kb in length
CRISPR components (Cas9 mRNA, guide RNA, and DNA oligos/ plasmids as template if desired) are injected into mouse zygotes, which are borne by surrogate mothers. Part of the new-borns will have incorporated the desired genetic modification at the targeted location.
Initiating a project
Model generation with either approach here begins with a discussion with us of your goals and determine the feasibility of your project. Contact our Laboratory Manager Ms. Heidi NG (Email: [email protected], Mobile: 6128 1647) to schedule an advisory meeting.
Microinjection service is an essential step in the process to achieve the following complex genetic modifications and mouse model creation:
Random DNA transgenesis
- Plasmid vector
- Bacterial artificial chromosome (BAC)
Foreign DNA is injected into fertilized mouse oocytes, which are borne by surrogate mothers. Part of the new-borns will have integrated the foreign DNA into their own genomes.
Endonuclease-mediated site-specific genome editing
- Knockout model by non-homologous end joining (NHEJ)
- Knock-in model mediated by homology directed repair (HDR) with a donor oligonucleotide/ plasmid
> Incorporation of point mutation (SNP)
> Insertion of a short fusion tag, such as HA or Flag
> Introduction of stop codon
> Creation of precise DNA deletion
> Insertion of loxP sites around one or more exons
> Swapping of the murine gene with its human ortholog
> Insertion of exogenous coding sequences encoding fluorescent proteins or Cre
Double-stranded plasmid donor DNA (Generally > 5 kb)
> Insertion of large fragment exceeding 5 kb in length
CRISPR components (Cas9 mRNA, guide RNA, and DNA oligos/ plasmids as template if desired) are injected into mouse zygotes, which are borne by surrogate mothers. Part of the new-borns will have incorporated the desired genetic modification at the targeted location.
Initiating a project
Model generation with either approach here begins with a discussion with us of your goals and determine the feasibility of your project. Contact our Laboratory Manager Ms. Heidi NG (Email: [email protected], Mobile: 6128 1647) to schedule an advisory meeting.
Pricing
Pronuclear/ cytoplasmic microinjection of at least 200 mouse zygotes followed by surgical transfer: HKD 15,000 per session **
Remarks:
** Charges for CRISPR-Cas9 reagents, DNA construct, heath screen, and animal transport are NOT included. It includes the cost of wild-type donor females at common genetic backgrounds, such as C57BL/6J and C57BL/6NJ (Up to 25 females).
Remarks:
** Charges for CRISPR-Cas9 reagents, DNA construct, heath screen, and animal transport are NOT included. It includes the cost of wild-type donor females at common genetic backgrounds, such as C57BL/6J and C57BL/6NJ (Up to 25 females).
Each session includes:
- superovulation of donor females of the desired host strain;
- setting up cross with background-matched stud males and checking plugs;
- collecting zygotes (one-cell fertilized embryos) at 0.5 dpc;
- performing pronuclear and/or cytoplasmic microinjection to deliver DNA, RNA, and/or ribonucleoproteins into the zygotes;
- washing the zygotes in accordance with guidelines promulgated by the International Embryo Technology Society (IETS) to eliminate the pathogens if necessary;
- preparation of pseudopregnant females;
- transfer of the surviving embryos into psuedopregnant recipients;
- housing for pseudopregnant females receiving embryo transfer and their offspring within the first 7 weeks (3 weeks gestation time and 4 weeks of post-natal growth);
- collection of tail biopsy from each mouse at weaning for genotyping analysis (PCR-based and sequencing-based verification); and
- delivery of tail biopsies to designated facility/ location for pick up by the investigator’s team.
Useful information
- There are several factors affecting the efficiency of GEM production. Since the design, quality and size of the RNA/DNA injected is of utmost importance for a successful outcome, NO guarantee can be given by LTCF for the number of live born mice or the number of positive transgenic/ mutant founders produced.
- The DNA construct or RNA submitted by researcher for microinjection should be prepared in recommended manner. Please contact us for the sample preparation protocols. The intactness, purity and concentration of the fragment to be injected should be assessed prior to submission.
- Preliminary studies on cultured cell lines are highly recommended to assess the integrity of the DNA construct and the function of the promoter or the cutting efficiency and specificity of the targeting sgRNA.
- If you require a different mouse strain other than C57BL/6J, we may be able to accommodate your needs. However, there is a surcharge for custom strains since transgenic efficiency is lower in most of the custom strains.
- Predicted founders at age of 5 weeks will be transferred to the investigator for breeding and validation of the transgender expression or desired genome edit in the N1 generation. Alternatively, breeding and screening of the N1 generation can be performed by LASEC on a fee-for-service basis.
- Additional session (Microinjection of ~100 mouse zygotes, HKD 5,000) will be available upon request at additional cost.
- A valid AEEC number must be provided prior to commencement of service. The strain name should be clearly specified in the AEEC project.
- Completed service request form should be returned to us prior to commencement of service.
- Our facility prioritises all service requests on a “first-come, first-serve” basis.