Bacterial Artificial Chromosome (BAC) Knock-in and Conditional Knock-In Rat Models

BAC Knock-in and Conditional Knock-In Rat Models
 
Bacterial Artificial Chromosomes (BACs) are ultra-low copy vectors that can hold up to 300 kb of genomic fragments, making them ideal vectors for introduction of entire genes including the regulatory regions into the rat genome. BAC transgenic rats are similar to other knock-in models in the expression pattern of the gene of interest, except that the BAC is randomly integrated in the genome as a transgene. Applied StemCell can create a BAC knock-in and conditional knock-in rat models for you.
 
Our services include:
  • BAC design and cloning strategy
  • Cloning and sequence validation of the BAC tailor-made to your specifications
  • Pronuclear microinjection of BAC into rat embryos
  • Genotyping
Alternatively, you can choose to have us do the BAC modification strategy and cloning, and you can do the pronuclear injections and transgenic rat screening yourself.
 
 Service Time
1.  DNA BAC vector design, cloning and recombineering  6-8weeks
Design, strategy, and BAC recombineering  
Enzyme digestion identification and sequencing to confirm
 
Purification of BAC DNA for microinjection
 
2. DNA pronuclear microinjection and implantation of embryos
 2 month
Pronuclear microinjection and embryo implantation
 7-10 weeks
3. Animal Care, Housing, and Genotyping Founders
1 month
Deliverable: Confirmed positive founder(s)
 
Case Studies
Case Study #1: Large DNA Fragment Insertion at a Neuronal Gene

Goal: To generate a transgenic rat model with a ChR2-GFP insertion (10.5 kb) at a neuronal gene of interest using BAC technology.

The donor DNA (10.5 kb) was microinjected into the pronucleus of Long Evans rat embryos. The pups born after microinjection were genotyped with a panel of PCR primers to detect the presence of the transgene sequence and further confirmed by Sanger sequencing. Three founder rats (F0) that were identified were bred with wild type Long Evans rats and twenty-two (22) F1 germline transmitted rats were born and delivered to customer. The results shown below are PCR screening data for F1 transgenic rats (Figure 1) that were confirmed by sequencing to have the gene of interest inserted at desired genomic locus.

CASESTUDY-Rat-BAC-KI-1

Figure 1. Identification of positive F1 transgenic rats. Twenty-two (22) F1 germline transmitted transgenic rats were identified using three sets of PCR primers (Lanes 1, 2, and 3) to identify transgene insertion at desired locus. PCR1 identified sequence at the 5’ integration site of donor construct (390 bp), PCR2 recognized sequence around the ChR2-GFP fragment and promoter (703 bp) and PCR3 recognized 3’ integration site at gene of interest (599 bp). These rats were also confirmed to have the transgene inserted by sequence analysis. Lane M: 100bp DNA marker. 

Case Study #2: Reporter Gene Insertion Rat Model

Goal: The purpose of this project is to generate a transgenic rat model with GFP insertion at a specific locus.

A BAC vector containing GFP inserted at the desired locus was prepared and microinjected into the pronucleus of embryos from Sprague Dawley rat strain. After F1 breeding, 6 out of 10 rats were identified as containing the BAC-GFP sequence at the desired locus sequence by PCR.

CASESTUDY-Rat-BAC-KI-2

Figure 2. (a) Schematic illustration of BAC-GFP construct and genotyping scheme; PCR reactions 1 to 4 are labeled in orange. (b) F1 rats from litter B (#2-6) were tested for PCRs 1, 3 and 4 and compared to F0 foudner; GeneRuler 100 bp DNA marker was used as standard.

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