Generation of Cloned Transgenic Cats Expressing Red Fluorescence Protein1

Abstract

A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 +/- 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.

Generation of red fluorescent protein transgenic dogs

“…We monitored the in vitro development and RFP expression in the canine reconstructed embryos. Red fluorescence was detectable in all stages of reconstructed embryos without sign of mosaicism, which corresponds with the studies of other species (Hyun et al, 2003;Lai et al, 2002b;Yin et al, 2007). The fluorescence seen in the reconstructed embryos at an early stage of embryogenesis (before the zygotic genome activation) is believed not to be derived from the transcription of the donor cell genome but from the cytoplasm of the donor cell, which contained RFP mRNA as well as RFP protein.…”

“…In this study, the mean efficiency of reaching pregnancy was 35.0% and of giving live birth was 1.7% per embryo transfer. These results are comparable to those obtained in other species, including pigs (Lai et al, 2002b;Park et al, 2002), calves (Bordignon et al, 2003;Forsberg et al, 2002), goats (Keefer et al, 2001), and cats (Yin et al, 2007). Moreover, these results are higher than average based on the previous reports using canine embryos reconstructed with nontransfected donor cells Kim et al, 2007;Lee et al, 2005).…”

“…The most difficult challenge in generating transgenic animal models in large animals has been in obtaining viable germ cells and embryonic stem cells (ESCs). However, recent successes in creating transgenic animals (Bordignon et al, 2003;Cibelli et al, 1998;Dai et al, 2002;Keefer et al, 2001;Schnieke et al, 1997;Yin et al, 2007) using somatic cell nuclear transfer (SCNT) gave promise to future generation of genetically modified models in large animal including dogs. Dogs (1) have organ sizes comparable to those of humans, (2) generally cohabitate with human beings, minimizing environmental effects, and (3) receive exceptional medical care (Mack, 2005;Ostrander et al, 2000;Sutter and Ostrander, 2004).…”

Application of Genetically Modified and Cloned Pigs in Translational Research

“…Genetically engineered animals including cell markers, such as fluorescent proteins, are highly useful in research that requires tracking of transplanted cells or tissues [55][56][57][58]. In rodents, transgenic animals expressing such cell markers as green fluorescent protein (GFP), red fluorescent protein (RFP) and β-galactosidase have been produced, making rodents useful in a wide range of research [57,[59][60][61][62][63].…”

The Effects of Manipulation Medium, Culture System and Recipient Cytoplast on In Vitro Development of Intraspecies and Intergeneric Felid Embryos

“…57: [385][386][387][388][389][390][391][392] 2011) is the world's first domestic cat (DC) cloned using an adult somatic cell as a donor nucleus [1]. To date, several reports have successfully documented domestic kittens [2][3][4][5] cloned by NT. There are many efforts currently underway to study the conservation of endangered felid species.…”