Cytomictic Anomalous Male Meiosis and 2nPollen Grain Formation inMertensia echioidesBenth. (Boraginaceae) from Kashmir Himalaya

PresentlyMertensia echioidesBenth. (Boraginaceae) collected from Kashmir Himalaya, India, is cytologically analyzed for the first time revealing2n=2x=24(diploid). Interestingly we found 4.3–6.2% syncytic meiocytes/PMCs with2n=4x=48(tetraploid) in addition to normal meiocytes (2n=24) during male meiosis. These comparatively larger PMCs (pollen mother cells) lead to the formation of fertile giant2npollen grains. A frequency of 6.4–13.3% PMCs shows transfer of chromatin material at prophase-I and, therefore, results in aneuploid meiocytes. Whole chromatin transfer by the process of cytomixis could also have led to the formation of tetraploid cells. Translocation heterozygosity is also evident in the form of multivalents in 12–17% diploid (2x) meiocytes at diakinesis and metaphase-I and is reported for the first time in this species. The syncytes formed depict open chain hexavalent and quadrivalent formation in the three populations with different frequencies. Moreover chromatin stickiness at metaphase-I is observed in 45% of PMCs in population-1 (P-1). Syncyte or unreduced PMC formation leading to unreduced fertile gametes is here speculated to act as a possible way out for infraspecific polyploidization in the species.

GENE INACTIVATION AND NONMEIOTIC ALLELE INTROGRESSION IN LIVESTOCK SPECIES USING TALENS

TALEN-induced double-strand breaks can be used for gene inactivation via repair by non-homologous end joining (NHEJ) or to stimulate homologous recombination (HR). HR can be used to introduce custom genetic modifications or to introgress naturally occurring alleles. We found that over 65% of custom-designed TALENs displayed activity in pig and cattle fibroblasts, with a typical percentage of indel positive chromosomes ranging from 20 to 45%. Isolation of individual clones with mono- and biallelic modifications to targeted loci was extremely efficient (up to 84 and 24% of clones, respectively) and could be accomplished without the aid of selection. Co-transfection of TALENs with a homologous repair template enabled precise insertion of a novel restriction site in nearly 40% of treated cells, with surprising levels of homozygosity. To prove that gene-edited Ossabaw swine cells were suitable for the generation of animals by cloning, we pooled colonies harboring both monoallelic and biallelic TALEN-induced frame-shift mutations in the swine low-density lipoprotein receptor (LDLR) and used them as nuclear donors for chromatin transfer. Pregnancy was established in 7/9 transfers, and 6 pregnancies were carried to term, resulting in the live birth of 18 piglets. Pigs heterozygous and homozygous for TALEN-induced mutations are being investigated as models of familial hypercholesterolemia (FH). We have additionally targeted the same locus for HR using a specified inactivating mutation. Fibroblasts heterozygous and homozygous for a specific 4-bp insertion into LDLR were created by allele introgression and have been cloned by chromatin transfer, demonstrating that gene editing can be used to create precise, swine knock-ins in a single generation. Allele introgression is also critical to livestock genetics, where crossbreeding has been a staple of breeding programs. Although major effect alleles for enhancing productivity and animal welfare have been discovered, the introgression of low-frequency alleles by traditional breeding is slow and inaccurate, involving recombination across the entire genome. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement in a diversity of livestock breeds. Co-transfection of a TALEN pair with a template containing a specific, naturally occurring allele was effective at the non-meiotic introgression of quantitative traits into the genome of cells from naïve cattle breeds, now being used to create founders by cloning. We will also present progress towards gene conversion by direct injection of livestock embryos. Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in up to 75% of embryos analysed, nearly half of which harbored biallelic modification. We will present alternative strategies for the incorporation of gene editing into livestock genetic improvement programs by either cloning or embryo treatment.

35 PRODUCTION OF CLONED MINIATURE CALVES USING CYTOPLASTS FROM COWS OF STANDARD SIZE

Numerous genetically similar cattle for use in research or teaching can be produced with chromatin transfer technology (Sullivan et al. 2004 Biol. Reprod. 70, 146–153). Miniature cattle can provide advantageous biotechnological models for teaching and study of human and animal diseases. Miniature cattle are approximately one fourth the size of standard cattle and, therefore, represent a potentially less expensive, safer, and more easily managed animal model. Our limited attempts to reproduce miniature cattle via in vivo and in vitro production of embryos resulted in poor response to superovulation and logistical challenges in recovering embryos or oocytes due to the small size of the animals. Thus, the purpose of this study was to evaluate the potential of embryos derived by chromatin transfer from fibroblasts of a miniature cow and cytoplasts from cows of standard size to produce viable offspring after transfer into recipient cows of standard size. The donor of somatic cells was a heifer that weighed 7.7 kg at birth. Chromatin transfer resulted in 19% (82/428) blastocyst formation. A total of 66 cloned blastocysts (65 excellent/good quality, 1 fair quality) were transferred into 26 synchronized recipients. While ultrasound revealed 13 pregnancies prior to 67 days of gestation, only 4 pregnancies of 5 fetuses were maintained beyond 100 days. Parturition was induced with dexamethasone and prostaglandin on Day 286 of gestation. One singleton (12.3 kg) and a set of twins (10.2 and 11.1 kg) were healthy at birth and normal at 1 week of age. Two fully developed singletons, weighing 21.4 and 13.6 kg, died in utero. The latter fetus exhibited a fixed dorsolateral deviation of the neck that complicated delivery despite a caudal obstetrical presentation. No abnormalities were noted in the size or structure of any placenta. Our results indicate that healthy miniature calves can be gestated by recipient cows of standard size after transfer of embryos derived by chromatin transfer. Unfortunately, the fetal wastage, fetal anomalies, and stillbirths observed with standard sized cattle also may occur.