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transgenics & microinjection - rat
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rat transgenics & microinjection

Service to generate transgenic mice by pronuclear microinjection (random gene insertion).

Transgenic mice are generated by microinjection of the linearized DNA cassette most commonly into the larger male pronucleus of the fertilized mouse zygote¹,²,³,⁴. In contrast to ES-cell gene targeting where the DNA is precisely integrated to a desired genome location by homologous recombination, the microinjected transgene inserts randomly and often contains head-to-tail directional copies known as concatomers⁵,⁶. Integration of the DNA cassette after the first karyokinesis results in a mosaic animal⁷ in which not all of its cells contain the transgene, so typically offspring are screened for the presence of the transgene by PCR genotyping methods. Those animals that test positive known as founder mice (F0) should be mated to determine germline transmission ratios. A detailed description of non-Mendelian germline transmission ratios of mice produced by transgenic construct microinjection is provided by Haruyama et al 2009⁸.  While in practice the generation of transgenic mice through microinjection is facile, the highly variable patterns of integration require the use of multiple transgenic lines and experimental validation. Validation methods such as nanopore sequencing to determine the transgene integration site(s)⁹ and copy number of the transgene array is available.
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model generation

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Catalog Nr

Service Description

Timeline

Deliverables

Pricing

Rat - Standard Pronuclear Microinjection 
RNMJ001
Rat Sprague Dawley strain transgene pronuclear microinjection
~7 - 8 weeks from time of microinjection
2 transgenic founders or 50 liveborn pups (if expression of the transgene would lead to embryonic or perinatal lethality) whichever comes first
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Rat - Premium Pronuclear Microinjection
RNMJ002
Premium service package - Rat Sprague Dawley strain transgene pronuclear microinjection

(includes design and construction of transgenic circuit, transgene gel purification, founder genotyping, breeding selected founders to test for germline transmission (F1's) of the transgene, determine approximate transgene array copy number). 
~2 - 3 months from time of microinjection
 2 transgenic founders or 50 liveborn pups (if expression of the transgene would lead to embryonic or perinatal lethality) whichever comes first
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Custom Rat strain - Standard Pronuclear Microinjection 
RNMJ003
Custom strain transgene pronuclear microinjection. Brown Norway, Fischer, Lewis, Long-Evans, Sprague Dawley, Wistar, Zucker 

Other custom strains available upon request
 ~7 - 8 weeks from time of microinjection
 2 transgenic founders or 50 liveborn pups (if expression of the transgene would lead to embryonic or perinatal lethality) whichever comes first
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Custom Rat strain - Premium Pronuclear Microinjection
RNMJ004
Premium service package - Sprague Dawley strain transgene pronuclear microinjection

(includes design and construction of transgenic circuit, transgene gel purification, founder genotyping, breeding selected founders to test for germline transmission (F1's) of the transgene, determine approximate transgene array copy number). 
~2 - 3 months from time of microinjection
2 transgenic founders or 50 liveborn pups (if expression of the transgene would lead to embryonic or perinatal lethality) whichever comes first
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support services

Service

Catalog Nr

Service Description

Timeline

Deliverables

Pricing

 Genotyping Assay Development - Transgenes
RNMJ005
NovoHelix offers a transgene genotyping service to help users develop robust genotyping protocols for screening animals after breeding and expansion of indidvidual transgenic founder lines.
PCR genotyping protocol
 Transgene Integration Site Mapping
RNMJ006
 Ultra-long read sequencing by nanopores to determine the integration site, approximate transgene copy number and basic structure of the transgene arrayed concatomers.  NovoHelix will extract high molecular weight genomic DNA, prepare the nanopore library with adaptors, and load onto the flow cell for nanopore sequencing. A bioinformatic summary of the transgene integration site, structure of the arrayed concatomers and approximate copy number is provided to the client for review. Assessement of the integrity of neighboring genes harboring the transgenic array is also provided. Additional coverage is ultra-long read coverage
 7-10 business days
A bioinformatic summary of the transgene integration site, structure of the arrayed concatomers and approximate copy number. Assessement of the integrity of neighboring genes harboring the transgenic array is also provided.  1.5-2x whole genome coverage from ultra-long reads of the assayed transgenic line
 Transgene vector construction
RNMJ007
Transgene vector construction and Sanger &/or Minion-sequence verified.

NovoHelix will provide an in silico map and provide restriction fingerprinting for plasmid structure via 3 digests. DNA is purified by a propriety AEX (anion exchange) chromotography and suitable for microinjection into mouse zygotes or nucleofection/electroporation.  The DNA is dialyzed on a membrane support in microinjection or electroporation buffer and then spun to remove particulate matter to obviate microinjection needle clogging.
 1 - 4 weeks depending on transgene vector complexity

5-10 micrograms of transgene plasmid; sequence validation, in silico map and 3  digests for restriction fingerprinting overall structure.
Transgene purification
 RNMJ008
 Purification of supercoiled plasmid by a proprietary anion exchange (AEX) chromatography, validation by restriction digestion and analysis by gel electrophoresis. Endotoxin levels are generally very low (0.05  – 0.5 ng LPS/µg) via our proprietary extraction method and are adequate for sensitive applications such as zygotic microinjection of mammalian embryos. The plasmid is predominantly in its supercoiled topology and free of RNA and protein contamination.  Linearization with a restriction endonuclease or with a homing nuclease such as PI-SceI, removal of vector backbone by gel extraction and drop dialysis in nucleofection/electroporation/microinjection buffer is included in the purification service. Validation by additional restriction digestions (greater than 3) is available with commensurate fee schedule.  Purification is compulsory to prevent the microinjection glass needle from clogging, to prevent embryo lysis and extensive delays in refitting and resetting the microinjection setup.
 transgene dsDNA donor vector construction - complex
 RNMJ009
Isogenic knockin dsDNA donor vector construction and Sanger-sequence verified with up to 20-kb insertion. For transgenes larger than 20-kb, please see our BAC transgenics services. NovoHelix will provide an in silico map and provide restriction fingerprinting for plasmid structure via 3 digests. DNA is purified by a propriety AEX (anion exchange) chromotography and suitable for microinjection into mouse zygotes or nucleofection/electroporation.  The DNA is dialyzed on a membrane support in microinjection or electroporation buffer and then spun to remove particulate matter to obviate microinjection needle clogging.
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rat colony husbandry & management 
RNMJ010
rat colony management including breeding, weaning, sampling, monitoring, per cage per day for the production of founders and F-generation pups
technology
references

1: Haruyama N, Cho A, Kulkarni AB. Overview: engineering transgenic constructs and mice. Curr Protoc Cell Biol. 2009 Mar;Chapter 19:Unit 19.10. doi:10.1002/0471143030.cb1910s42. Review. PubMed PMID: 19283728; PubMed Central PMCID: PMC2743315.

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