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Strain Development

Techniques to develop rat models

F1, F2 and their reciprocal crosses:
For the F1 generation, 1 male SHRSP was mated with two WKY females, 3 F1 males were mated with 6 F1 females (brother x sister) to generate the F2 cross. For the reciprocal crosses 1 male WKY was mated with two SHRSP females, 3 F1 males were mated with 6 F1 females (brother x sister) to generate the F2 cross. RGD ID: 4145662

Using Zinc-Finger Nucleases to Generate Gene-Specific Mutated Rats:
Engineered zinc-finger nucleases induce site-specific, double-stranded DNA breaks that on repair result in a targeted mutation. This is done by combining ZFN-encoding nucleic acids, then testing them in cultured cells. These are microinjected into one-cell embryos and then are transmitted into the germline and then backcrossed to generate multiple strains with unique mutated alleles. RGD ID: 4131263

ENU mutants:
Mu transpososome is used for the in vitro cleavage of the mismatch target site which is a complex of MuA transposase and Mu-end DNA, this is followed by rejoining of the target site to the 3’ ends of the Mu-end DNA, followed by screening to detect single nucleotide substitutions within the targeted gene. RGD ID: 4239490
This procedure involves injecting a split dose of ENU (2 x 40 mg/kg)  into males who are then mated (10 weeks after the second injection). DNA and sperm from the offspring are collected and screened. If a successful mutation is detected then intracytoplasmic sperm injection (ICSI) is used to derive offspring. (from Rat ENU mutagenesis NBRP)

Conplastic Strains:
These can be developed by backcrossing the mitochondrial genome from the donor onto the nuclear genetic backround of the recipient strain this results in selective replacement of the mitochondrial genome of SHR with the mitochondrial genome of BN to create a conplastic SHR model . The conplastic strain is genetically similar to the parent strain except for the difference in the mitochondrial genome. RGD ID: 1627567

Congenic strains:
A segment of a chromosome from a strain (donor) is introgressed into the genetic background of another strain (recipient). This is done by mating the first strain to the second and then backcrossing to the second strain multiple times. To maintain the heterozygosity for the region of interest the offspring are genotyped using markers that flank the region of interest. RGD ID: 61051
Strategies to speed-up the process of deriving congenics:
1. Marker assisted derivation
2. Supersonic derivation:  in each backcross generation obtain embryos from 4-week-old superovulated females, mated with parental males and transferred into pseudo-pregnant foster mothers.

Consomic strains:
Parental strains A and B are crossed to get the heterozygous F1 generation, which is then backcrossed to the parental strain B. The backcrossed offspring (N2) are genotyped and the heterozygous offspring along the target chromosome are further backcrossed for 4-8 generations. This gives offspring that are isogenic strain B background for all chromosomes except the target chromosome. Selected animals are then brother x sister mated to get offspring that are homozygous for the chromosome of interest. RGD ID: 1303384

CRISPR/Cas9 mutants:
CRISPR/Cas system is composed of a Cas9 nuclease and a guide RNA that directs the nuclease to the target DNA through base-pairing rules (RGD ID: 11070050). This procedure can generate rat strains that carry mutations in multiple genes by directly injecting the edited RNAs into single-cell embryos. The mutation in the F1 generation can be validated using PCR. RGD ID: 10045593



RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.