The polyadenylate tail of eukaryotic mRNAs is thought to influence various metabolic phenomena including mRNA stability^ translation initiation, and nucleo-cytoplasmic transport. We have analyzed the fate of mRNAs following inactivation of poly(A) polymerase in Saccharomyces cerevisiae containing a temperature-sensitive, lethal mutation {papl-1) in the gene for poly (

Introduction: Recombinant factor VIII (rFVIII) is presently produced and purified from mammalian cell lines in liquid culture but has proven to be costly. The milk of transgenic livestock can yield an abundant source of complex therapeutic proteins. While properly processed recombinant factor IX and protein C have been produced at levels up to 100 U/ml in the milk of transgenic pigs, levels of full length rFVIII were <1 U/ml, likely due to secretory inefficiency, chain dissociation and proteolytic degradation. Methods: Transgenic mice provide both insight into the encoding fidelity of the transgene constructions as generally expressed by mammary epithelia and useful amounts of posttranslationally modified, recombinant protein for evaluation. A rFVIII variant bioengineered for enhanced secretion (F309S/226aa/N6) was expressed in the milk of transgenic mice using the 4.2 Kbmurine Whey Acidic Milk Protein (mWAP) 5'-promoter & 1.3 kb of mWAP 3'-UTR elements. Transgenic mice were made containing F309S/226aa/N6 in combination with von Willebrand factor (vWF) and/or alpha1-antitrypsin (AAT) to help stabilize the rFVIII and reduce proteolytic degradation. Results: Pronuclear injection of equimolar mixtures of the transgenes was used to produce multigenic mice. 18 founder lineages were outbred with wild-type mice. Milk from F1 lineages was screened for rFVIII by ELISA; 0 of 4 F1 lineages bigenic for FVIII&vWF expressed FVIII, 2 of 6 F1 lineages bigenic for FVIII&AAT expressed rFVIII at a range of 1-11 ug/ml, while 1 of 4 F1 trigenic lineages expressed rFVIII at 61 ug/ml. Western blots showed primarily intact rFVIII that quantitatively agreed with ELISA results. Only the milk of the trigenic FVIII-AAT-vWF mice showed high level coagulation activity (34 U/ml). Conclusions: Very high level rFVIII milk-specific expression can be achieved using high secretion efficiency FVIII variants when co-expressed with vWF and AAT. This multi-gene strategy is being studied in cloned transgenic pigs.

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The 2‐micron plasmid is a small genetic element that persists in Saccharomyces cerevisiae with chromosome like stability. It encodes for four proteins (Rep1, Rep2, Raf1, and Flp) that appear to provide no advantage for the host cell, but instead have a role in replicating and maintaining a steady state concentration of the plasmid‐‐ 1. Flp recombinase is known to promote amplification of the 2‐micron; disruption of the FLP gene is a valid way of preventing the necessary amplification of the plasmid to pass itself on. The 2‐micron is a selfish DNA element that is able to maintain a copy number of 40–60, however its ability to maintain these levels is not completely understood. To study this regulation we have established a quantitative and efficient means to monitor copy number. We used CRISPR‐Cas9 to replace ADE2 (encoded on 2‐micron/ADE22) with mCherry, enabling copy number to be monitored using flow cytometry. Preparation of the strains required no additional staining, allowing for quick processing and analysis. To establish the sensitivity of the system, mCherry expression was monitored in strains that had ectopic expression of the flp‐H305L mutant, which destabilizes the 2‐micron plasmid 2. We observed a decrease in mCherry expression in these strains, consistent with the role Flp plays in 2‐micron amplification. This reduction in copy number was confirmed using quantitative PCR to examine expression of mCherry from the 2‐micron. Current studies are employing this system to monitor how copy number is affected by two long non‐coding RNAs (lncRNA), which are expressed from the plasmid and regulated by Rrp6p and the TRAMP complex, components of the nuclear RNA surveillance pathway. Support or Funding Information SJFC internal funds K.-M.ChanY.-T.LiuC.-H.MaM.JayaramS.SauPlasmid701217E. L.TsalikM. R.GartenbergYeast14847852