Compositions and methods for generating transgenic animals
A transgenic animal, transgenic technology, applied in biochemical equipment and methods, botany equipment and methods, genetic engineering, etc.
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Embodiment 2
[0092] Example 2 - Altering Gene Expression in Embryos
[0093] In the first set of experiments, the vector was DNA containing a CMV (cytomegalovirus)-promoter driving expression of DsRed2, and a second promoter, U6, driving hairpin DNA expression, was injected into the tail vein of mice, which Hairpin DNA is targeted for reduced Bmp4 expression. In this set of experiments, all embryonic cells fluoresced indicating that the construct had been taken up and expressed in those cells. The chance of false positives is eliminated by placing the marker and DNA in the same frame, where the marker is expressed but the hairpin DNA is not.
[0094] Since the hairpin DNA is in the same frame as the marker, and all cells have the marker, there is no reason to think that Bmp4 is not reduced either. This predictability has been confirmed in 9 independent DNA. In each case, using published techniques, markers were found in all cells of embryos or newborns developing from embryos. In all c...
Embodiment 3
[0099] Example 3 - Injection and expression of the neomycin gene in pregnant mice and their offspring
[0100] This experiment was performed to test whether mice could be injected with a foreign gene sequence that would then be expressed in the following generations of offspring. Female mice, on days 6-6.5 of gestation, were injected at or near the tail vein with a plasmid expression vector encoding the neomycin antibiotic resistance gene.
[0101] A suitable dosing regimen involves dissolving 20 micrograms of vector DNA in sterile phosphate-buffered saline (PBS) or Ringer's solution and injecting the solution into the animal. A suitable carrier concentration is 0.1 micrograms per microliter (eg, use a volume of 200 microliters to dissolve 20 micrograms of carrier). The injection rate can be slow, over a period of 20 to 30 seconds. Higher or lower dosages can be used.
[0102] Neomycin mRNA expression was analyzed in parental animals and their offspring, which were egg cyli...
Embodiment 4
[0105] Example 4 - Injection and expression of GFP and ampicillin genes in pregnant mice and their offspring
[0106] As in Example 3, female mice were injected with plasmid expression vectors encoding green fluorescent protein (GFP) and ampicillin antibiotic resistance genes on day 6-6.5 of pregnancy. Genomic DNA from the resulting offspring was analyzed for the presence of GFP and ampicillin DNA using polymerase chain reaction (PCR). DNA from a total of 35 offspring (male and female) from 5 littermates was tested for the presence of GFP and ampicillin resistance (ampr) gene sequences by PCR. Of these mice, one or more animals from each litter, and a total of 12 animals were positive for the GFP and ampr sequences. By sequencing the PCR products, it was confirmed that the PCR products (520 bases for GFP and 534 bases for the ampicillin resistance gene) were PCR products of GFP and ampr.
[0107] The offspring animal pairs are then mated to produce second generation (F1) ani...
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