Molecular Genetics and Engineering

The MBP utilizes a variety of different techniques including endonuclease (CRISPR) gene editing, BAC recombineering, Yeast Recombineering, Gibson Assembly Cloning, and traditional ligation to create the desired Knockout (KO), Conditional Knockout (cKO), Knockin (KI) and Transgenic (Tg) vectors in the most efficient manner possible. A client need only provide the gene name and the desired product, and our knowledgeable staff will take care of the rest.

CRISPR Gene Editing

The MBP offers CRISPR guide design specific to desired mutations including InDel (insertion/deletion) Knockout, small cassette Knockin (ssODN repair template utilizing homology directed repair (HDR) pathways). Use of long ssODN (ssDNA) for larger Knockin (cKO or reporter knockins in addition to plasmid based repair of larger fragments up to 10kb). We design and target either zygotes or ES cells depending on the allele and project scope.

Off target analysis is offered for any potential off targets of concern. The MBP designs guides to avoid potential off targets but with particular allele or project requirements, it may still be necessary or prudent to screen for and if needed, breed out non desired off targets.

Construct Creation

Our standard vector construction service fee includes:

Project consultation with our team of scientific experts
Design for mutant model (from our client, we only need a gene name and mutant model type – i.e. KO, KI, transgenic, etc.)
Thorough informatics review, including the verification of exon/intron structure by blasting the cDNA against the mouse genome and confirming the coding region using virtual translation tools.
Construct Maps and sequence files in Vector NTI, genbank, and word.
Purified plasmid DNA from Final Construct (at least 20 ul of a 50 ng/ul solution).
Raw sequencing data of final construct
Figure-usable schematic

Upon request, we can also provide:

Methods section
Glycerol stock of final plasmid
Glycerol stock of parent BAC clone
Gel images of:
- PCR verification steps
- Restriction Verification steps

Construct Verification

For final construct verification, we perform 5-6 restriction digests and complete sequencing across all PCR amplified regions and over all insert junctions. Design plan, construct sequence file and figure-usable schematic and description are sent to the client for approval prior to beginning vector construction service work. All service work is billed upon milestone completion. Unlike other recombineering methods, we perform restriction analyses verification at most steps of vector construction. This results in increased speed and reliability and allows us to build conventional and conditional KO constructs in as little as 8-12 weeks.

For additional information on BAC Recombineering and Targeting Schematic’s please view the links below:

Targeting Vector Verification (KOMP/EUCOMM)

KOMP/EUCOMM construct verification via restriction digestion analysis and construct preparation for electroporation.

This service includes ordering a KOMP vector glycerol stock (or receiving a EUCOMM purified plasmid ordered by the client), retransforming and purifying plasmid DNA from bacterial culture, and restriction digestion verification using at least 5 restriction enzymes, linearization and purification for electroporation. Additional PCR may be performed in order to identify correct clones.

Transgenic Vector Purification (BAC/High Copy Plasmid)

Final purification of transgenic vectors prior to Pronuclear injection to create transgenic founders.

For high copy plasmid transgenic vectors services, we include the restriction digestion, gel purification to remove prokaryotic vector sequence and final purification of transgenic fragment prior to pronuclear microinjection to create transgenic founders.

For BAC transgenics services, we include the isolation of BAC DNA for bacterial culture and final purification prior to injection including PFGE analysis of DNA integrity.