Lesley Crocker

Cdk6-cyclin D3 activity in murine ES cells is resistant to inhibition by p16(INK4a).

Through a screen aimed at identifying genes that are specifically upregulated in embryomic stem (ES) cells but not primitive ectoderm, we identified cyclin D3. This was surprising since cyclin D activity is generally believed to be inactive in ES cells even though retinoblastoma tumor suppressor protein (pRb) accumulates in a predominantly hyperphosphorylated state. Cdk6 is the major catalytic partner for cyclin D3 in ES cells and exhibits robust pRb kinase activity that is downregulated during the early stages of ES embryoid body differentiation. To investigate the basis underlying the insensitivity of ES cells to ectopic p16 expression, we show that Cdk6–cyclin D3 complexes are not subject to inhibition by p16, similar to Cdk–viral cyclin complexes. These observations show that specificity exists between Cdk4/6–cyclin D complexes and their ability to be targeted by p16. Our data suggest that Cdk6–cyclin D3 activity in other cell types, including tumors, may also be refractory to p16-mediated growth inhibition and raises the possibility of additional specificity within the INK4 family.

Efficient Generation of α(1,3) Galactosyltransferase Knockout Porcine Fetal Fibroblasts for Nuclear Transfer

Pigs are currently considered the most likely source of organs for human xenotransplantation because of anatomical and physiological similarities to humans, and the relative ease with which they can be bred in large numbers. A severe form of rejection known as hyperacute rejection has been the major barrier to the use of xenografts. Generating transgenic pigs for organ transplantation is likely to involve precise genetic manipulation to ablate the α(1,3) galactosyltransferase (galT) gene. In contrast to the mouse, homologous recombination in livestock species to ablate genes is hampered by the inability to isolate functional embryonic stem cells. However, nuclear transfer using genetically targeted cultured somatic cells provides an alternative means to producing pigs deficient for galT. In this study we successfully produced galT+/− somatic porcine fetal fibroblasts using two approaches; positive negative selection (PNS) using an isogenic targeting construct, and with a promoterless vector using non-isogenic DNA.

In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts.

The in vitro development of porcine nuclear transfer embryos constructed using primary cultures from day 25 fetal fibroblasts which were either rapidly dividing (cycling) or had their cell‐cycle synchronized in G0/G1 using serum starvation (serum‐starved) was examined. Oocyte‐karyoplast complexes were fused and activated simultaneously and then cultured in vitro for seven days to assess development. Fusion rates were not different for either cell population. The proportion of reconstructed embryos that cleaved was higher in the cycling group compared to the serum‐starved group (79 vs. 56% respectively; P < 0.05). Development to the 4‐cell stage was not different using either population. Both treatments supported similar rates of development to the morula (1.5 vs. 7%, cycling vs. serum‐starved) and blastocyst stage (1.5 vs. 3%, cycling vs. serum‐starved). The blastocyst produced using cycling cells had a total cell number of 10. Total cell numbers for the three blastocysts produced serum‐starved cells were 22, 24, and 33. These blastocysts had inner cell mass numbers of 0, 15, and 4, respectively. Six hundred and thirty‐five nuclear transfer embryos reconstructed using serum‐starved cells were transferred to 15 temporarily mated recipients for 3–4 days. Of these, 486 were recovered (77% recovery rate) of which 106 (22%) had developed to the 4‐cell stage or later. These were transferred to a total of 15 recipients which were either unmated or mated. Seven recipients farrowed a total of 51 piglets. Microsatellite analysis revealed that none of these were derived from the nuclear transfer embryos transferred. Mol. Reprod. Dev. 57:262–269, 2000.

SECRETION OF EUKARYOTIC GROWTH HORMONES IN ESCHERICHIA COLI IS INFLUENCED BY THE SEQUENCE OF THE MATURE PROTEINS

We report the construction of secretion plasmids expressing the fusion proteins, OmpA::pGH (pSpGH.01) and OmpA::hGH (phGH.01), and compare the secretion of mature porcine growth hormone (pGH) and human growth hormone (hGH) employing Escherichia coli. E. coli [phGH.01] secreted 10-15 micrograms hGH/ml/A600 cells into the periplasmic space, representing 30% of total periplasmic proteins. E. coli [pSpGH.01], however, secreted 30-fold less mature pGH. On the basis that both pSpGH.01 and phGH.01 are stably maintained in E. coli and in vitro transcription/translation data showed equivalent expression of OmpA::pGH and OmpA::hGH precursors, we attribute the higher secretion of hGH to the translocation-competent OmpA::hGH protein configuration. Two OmpA::GHF (growth hormone fusion) precursors, OmpA::GHF.02 and OmpA::GHF.03, both with hGH helix 3/helix 4 together instead of the pGH equivalent, secreted mature proteins as efficiently as OmpA::hGH. We propose that hGH helices 3 and 4 in these OmpA::GHF precursors play a major role in the folding of the precursor to a translocation-competent state, mimicking the translocation-competent nature of the OmpA::hGH precursor.

Complete sequence of a hair-like intermediate filament type II keratin gene.

The Intermediate Filament (IF) superfamily comprises several multigene families, of which the two keratin families are the largest. The keratin IF genes are expressed in epithelial tissues in differentiation-specific patterns and recently we reported the sequence and expression of a hair IF type II keratin gene (KRT2.9). Two related genes were present in the cosmid containing KRT2.9 and we have now sequenced one of them and found that it encodes a hair-like IF type II protein (KRT2.13). However, KRT2.13 is not expressed in the hair follicle. Interestingly there is significant sequence homology between introns 1, 5 and 6 of KRT2.13 and KRT2.9 to suggest gene conversion of these regions or possibly conservation of functional sequences.

Keratin Gene Expression in Wool Fibre Development

Hair growth has been studied for a long time and many of the keratin proteins of wool were sequenced over a decade ago, yet little is known concerning the coordination of expression of the individual keratin genes and the gene families, or of the molecular signals which are involved. With a view to addressing these questions we are using the wool keratin IF type II gene family as a model for keratin gene expression during fibre development