Method of Genetically Altering and Producing Allergy Free Cats
a technology of genetic modification and cat, applied in the field of transgenic cats, can solve the problems of time involved, unappealing treatment for allergy sufferers, and high cost of regular allergy
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example 1
Construction of Targeting Vector
A. Homology Arms
[0268]The DNA sequence of a continuous stretch of 22182 base pairs including and surrounding chains 1 and 2 of Fel d I was isolated and identified. The promoter sequence for each chain, and found that both chains share this promoter. Chains 1 and 2 are encoded on complimentary DNA strands, and transcribed in opposite directions (FIG. 7). Additionally, sequences downstream of the 3′ end of the genes of chains 1 and 2 were identified.
[0269]The assembly of the sequence data containing Fel d I (i) chain 1, (ii) chain 2, (iii) promoter and (iv) the downstream homology arms was performed using Polymerase Chain Reaction (PCR) amplification and DNA sequencing. The published polynucleotide sequence for the Fel d I gene (Griffith et al., Gene, 113(2):263-8, 1992, and Morgenstern et al., Proc. Nat'l. Acad. Sci. USA, 88:9690, 1991), was used as a starting point for a variation of thermal asymmetric interlaced PCR (TAIL-PCR) described in Liu & Whit...
example 2
EMBRYONIC FIBROBLAST CELL LINES
[0284]Somatic cat cell lines have been established which provide a viable alternative to cat embryonic stem cells for the purpose of targeting the Fel d I gene.
A. Derivation of Cat Somatic Cell Lines-Feline Embryonic Fibroblasts FEFs
[0285]1. Fetal tissue was isolated from the uterine horns of pregnant queens by standard ovariohysterectomy between 3 and 6 weeks of gestation. Extra-embryonic membranes were dissected away from the fetal tissue.
[0286]2. Connective tissue was then isolated, minced, and incubated in a basal medium of DMEM containing 2 mg / ml collagenase and 0.25% trypsin at 37 degrees Celsius for 15 minutes with periodic agitation
[0287]3. Embryonic fibroblasts were separated from remaining undigested tissues by straining through a 50 micron cell, rinsing with PBS and concentrating by centrifugation.
B. Culture and Expansion of Feline Embryonic Fibroblasts
[0288]Primary cat somatic cells were successfully cultured, expanded and frozen following ...
example 3
Targeting of Embryonic Somatic Cell Line
[0292]Gene targeting in somatic cells has enjoyed years of successes in many different species. We use the vector described above to target Fel d 1 in the feline fibroblast cell line
[0293]Cat embryonic somatic cell lines (FEFs) are electroporated with exogenous DNA targeting constructs, as described for cat embryonic stem cells above.
B. Selection
[0294]1. Positive Selection of FEFs with Gentamicin (G418): FEF cells that retained the neomycin resistance gene (Neo) survived, when cultured in 500 micrograms of G418 per ml of ES-DMEM, 24 hours after electroporation. Whereas, FEFs without the Neo gene died within 2-7 days of culture in G418.
[0295]Negative Selection of FEFs with Ganciclovir: FEFs that retained the HSV-1 tk gene 24 hours after electroporation died within 2-7 days of subsequent culture in ES-DMEM medium containing 500 nanograms of ganciclovir. Whereas, FEF cells that lacked the HSV-1 tk gene survived culture in gancic...
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